US010208322B2 (12 ) United States Patent ( 10) Patent No. : US 10 ,208 , 322 B2 Coelho et al. (45 ) Date of Patent: * Feb . 19, 2019 (54 ) IN VIVO AND IN VITRO OLEFIN ( 56 ) References Cited CYCLOPROPANATION CATALYZED BY HEME ENZYMES U . S . PATENT DOCUMENTS 3 , 965 ,204 A 6 / 1976 Lukas et al. (71 ) Applicant: California Institute of Technology , 4 , 243 ,819 A 1 / 1981 Henrick Pasadena , CA (US ) 5 ,296 , 595 A 3 / 1994 Doyle 5 ,703 ,246 A 12 / 1997 Aggarwal et al. 7 , 226 , 768 B2 6 / 2007 Farinas et al. ( 72 ) Inventors : Pedro S . Coelho , Los Angeles, CA 7 , 267 ,949 B2 9 / 2007 Richards et al. (US ) ; Eric M . Brustad , Durham , NC 7 ,625 ,642 B2 12 / 2009 Matsutani et al. (US ) ; Frances H . Arnold , La Canada , 7 ,662 ,969 B2 2/ 2010 Doyle et al. CA (US ) ; Zhan Wang , San Jose , CA 7 ,863 ,030 B2 1 / 2011 Arnold (US ) ; Jared C . Lewis , Chicago , IL 8 ,247 ,430 B2 8 / 2012 Yuan 8 , 993 , 262 B2 * 3 / 2015 Coelho ...... • * • C12P 7 /62 (US ) 435 / 119 9 ,399 ,762 B26 / 2016 Farwell et al . (73 ) Assignee : California Institute of Technology , 9 , 493 ,799 B2 * 11 /2016 Coelho ...... C12P 7162 Pasadena , CA (US ) 2006 / 0030718 AL 2 / 2006 Zhang et al. 2006 /0111347 A1 5 / 2006 Askew , Jr . et al. 2007 /0276013 AL 11 /2007 Ebbinghaus et al . ( * ) Notice: Subject to any disclaimer , the term of this 2009 /0238790 A2 9 /2009 Sommadosi et al. patent is extended or adjusted under 35 2010 / 0056806 A1 3 / 2010 Warren U . S . C . 154 (b ) by 0 days . 2010 /0168463 A1 7 /2010 Hirata et al. This patent is subject to a terminal dis 2010 /0240106 AL 9 /2010 Wong et al . claimer . 2011/ 0196086 A18 / 2011 Matsushita et al. ( Continued ) (21 ) Appl. No. : 15 /278 , 561 FOREIGN PATENT DOCUMENTS (22 ) Filed : Sep . 28, 2016 EP 0 200 638 B1 4 / 1986 (65 ) Prior Publication Data WO 2007 / 144599 A2 12 / 2007 US 2017 / 0247725 A1 Aug. 31 , 2017 (Continued ) OTHER PUBLICATIONS Related U . S . Application Data Adams, P . et al ., “ Phenix : a comprehensive Python -based system for (63 ) Continuation of application No . 14 /625 , 449, filed on macromolecular structure solution ,” Acta Crystallogr ., Sect . D , Feb . 18 , 2015 , now Pat . No . 9 ,493 ,799 , which is a Biol. Crystallogr ., 2010 , D66 ( 2 ) : 213 -221 . continuation of application No. 14 / 185 , 861, filed on ( Continued ) Feb . 20 , 2014 , now Pat. No . 8 , 993 , 262, which is a continuation of application No . PCT/ US2013 / 063577 , filed on Oct . 4 , 2013 . Primary Examiner — Pancham Bakshi (74 ) Attorney, Agent, or Firm — Kilpatrick Townsend & (60 ) Provisional application No . 61/ 711 ,640 , filed on Oct. Stockton LLP 9 , 2012 , provisional application No . 61 /740 , 247 , filed on Dec . 20 , 2012 , provisional application No . ABSTRACT 61 /784 ,917 , filed on Mar . 14 , 2013 , provisional (57 ) application No . 61/ 838 , 167 , filed on Jun . 21, 2013 , The present invention provides methods for catalyzing the provisional application No. 61/ 815 ,997 , filed on Apr. conversion of an olefin to any compound containing one or 25 , 2013 , provisional application No . 61/ 818 , 329 , more cyclopropane functional groups using heme enzymes . filed on May 1, 2013 , provisional application No . In certain aspects , the present invention provides a method 61/ 856 ,493 , filed on Jul. 19 , 2013 . for producing a cyclopropanation product comprising pro (51 ) Int. CI. viding an olefinic substrate , a diazo reagent, and a heme C12P 13 / 02 ( 2006 .01 ) enzyme; and admixing the components in a reaction for a C12P 7762 ( 2006 . 01 ) time sufficient to produce a cyclopropanation product. In C12N 9 /02 ( 2006 . 01 ) other aspects , the present invention provides heme enzymes (52 ) U . S . CI. including variants and fragments thereof that are capable of CPC ...... C12P 13 /02 (2013 . 01 ) ; C12N 9 / 0004 carrying out in vivo and in vitro olefin cyclopropanation (2013 .01 ); C12N 9 /0042 (2013 . 01 ) ; C12P 7762 reactions. Expression vectors and host cells expressing the ( 2013 . 01 ) ; C12Y 106 / 02004 ( 2013 .01 ) ; YO2P heme enzymes are also provided by the present invention . 20 /52 (2015 . 11 ) (58 ) Field of Classification Search CPC ...... C12P 13 /02 , C12P 7 /62 ; C12N 9/ 0042 7 Claims, 46 Drawing Sheetsts See application file for complete search history. Specification includes a Sequence Listing . US 10 , 208 , 322 B2 Page 2

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FIG . 13 2 mM glucose ABC Ph . Ph + EDA + OD variable 5 % MOH 500 ul . COOEL 20 mM 10 mm 2 hrs

0 . 900 WANAWAWA 0 .800 40 0. 700 Mp38/jouw) 0 .600 2 . 0 , 500 50. 400 20. 300 munnmannnnnnnnnMAKINARAK HANAKAKKUK ** * * * * * * * * * * wwwwwwwwwwwwwwww ------0 ,200 0 . 100 Een 0 .000 10 20 30 40 50 60 Cell density (OD 600nm )

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2 MM glucose COOEE 2411BME- CIS off X whole - cells 5 % MeOH [varlable ) 10 mM OD : 00 - 22 2 hrs 50 . 0 45 . 0 * 35 . 0 w ?? 30 .0 25 . 0 20 . 0 nafanana como 0 10 20 30 40 50 60 ( styrene] (MM )

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Phth EDA + ABC OD - 25 5 % MeOH COOEt 20 mM 10 mm OD = 25500 UL 80 .0 70 .0 wwwWMA 60 . 0

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R3=halide,ORNR2R R4=halide,ORNR2R pyrethrin1 R=H,alkyiarylalkenyi R2=H,alkylaryiOR B(OR)2,SIR noranthoplone NYEST AH

AS anthop?alone cost RR mn P450 Cyclopropanation EDA Wh P450 cyclopropanation EDA geranylacetone

2 2,4-diene IN ER 2,5-dimethylhexa 1 FIG.17D FIG.17A FIG.17B FIG.17C U . S . Patent Feb . 19 , 2019 Sheet 18 of 46 US 10 ,208 , 322 B2

ONSHOVA N BM3- CIS BM3- CIS Absorbance C400S TAWA Sobota w wwg pengepoole 000ODUKOWSKOCOGORO soppogodnotenie K o or WOODOO WAKAWODACHUNKO W SKIGOMA WOKA UX0000 350 400 450 500 550 600 650 700 Wavelength (nm )

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E FIG . 19A HO dithionite Phe ST styrene E NAD( P ) H Scys Eto , Na E?' Fe - Cys < E?NAD (P ) H EDA

of E NAD ( P )H | Ph “ co?E: Oser E°' Fe - Ser > E?NAD( P ) H X = Scys, Oser FIG . 19B T438 37V T268A SA328

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COOEE 10 MM Na2S204 P o sition au P450 0 . 7 M KPI PH 8 . 0 Phone COOEt 5 % MOH 15 UM cis 30 MM ( 0 . 15 % mol eg ) wrt EDA PANTO Phone CH20001 styrene oxide trans not formed when Na2S204 is the reductant

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SAs COOE : 5 % Meon PT COOEI PagulastCOOEI whole cells RAS 20 mM 10 mM OD600 = 25 variable Phone COOET Ph? 00E cis trans

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Whole . Glucose | Yield Total Effect of cis : " ee Pee (IM ) ( % ) * Turnover glucose density trans * trans Catalyst (mmol gw ") addition ( % ) (Ecdw L ') BM3 0 .120 + 0 . 03 5 . 53 34 : - 73 -22 CIS 60 (P4503M3 - CIS ) BM3 2 13 0 .240 + 0 .02 1 - 98 5 .53 48 : - 86 - 30 CIS 52 (P4503413 MAU - CIS ) wwwwwww ABC 0 35 0 .550 + 0. 06 6 .38 70 : -95 - 11 CIS 30 ORO ( P411FM - CIS ) 2 1 48 0 . 760 0 .01 + 37 6 . 38 76 : 1 - 961 - 14 ABC 24 ( P411BM3 - CIS ) VANNKAKUNA * Based on EDA . | Diastereomeric ratiosTURNE and enantiomeric excess were determined by CC analysis . * (2R , IS) -- ( 2S , 1R ). $ (2R , 1R ) - (2S , 19 ). FIG . 29 U . S . Patent Feb . 19 , 2019 Sheet 30 of 46 US 10 ,208 ,322 B2

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E . coli whole cells Ph M2 M9 Phnom COOEt 5 % MeOH 20 mM 10 mm

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FIG . 31 U . S . Patent Feb . 19 , 2019 Sheet 32 of 46 US 10, 208 ,322 B2

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ONURROR WWWWWWWWWWWWWWWWWW A AANAA Catalyst Yield ( % ) * | cisztranst % ee cist % ee transs ABC - CIS 60 :40 WWW -93 (P4113M3 - CIS ) ABC - CIS 16 17 : 83 (P411FMZ - CIS ) no induction VAMANATARA ARANANMURUNKA HAMAM Empty pcWori 15 11 :89 ABC - CIS 13 46 :54 (P411 BMZ- CIS ) + dithionite W ERKCREW ULAN * Based on EDA . † Diastereomeric ratios and enantiomeric excess were determined by GC analysis , * (2R , 15 ) - ( 2S ,1R ) . $ ( 2R1R ) - ( 25 , 19 )

FIG . 32 U . S . Patent Feb . 19 , 2019 Sheet 33 of 46 US 10 , 208 , 322 B2

2 mM glucose COOEE M9 Ph o ol ABC - CIS whole - cells 5 % MeOH Ph " COOEt 500 uL 20 mM 10 mM OD600 variable 2 hrs

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FIG . 35A native non - natural monooxygeneration P4508M3 cyclopropanation

dithionite O2 persone EO ! Niihiopite men efficient reductionw wo E'NAD (P)HÍ RI NAD(P )H + 02 poorreduction N EIOOG N2 Elfe - Cys < E NAD (P ) ( H ) P4118M3 pamil Atelier EO NADPH N . N efficient reduction Rand COOEt Ser X - S , O EO Ser EO NAD (P )( H )

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MINE NH,NELAY Ph.^ femme PhA, NHANH, we PhA Milnacipran

150°C A NzL MRKAWRRRRRR H !NE

N Ph.^

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FIG.36A FIG.36B FIG.36C U . S . Patent Feb . 19 , 2019 Sheet 37 of 46 US 10 ,208 ,322 B2

LDAthenHCI 0oMe MeOH K2CO3 0720" Ph.A 07

)ee%88(auxilliary Rhy(4S-MEAZ)4 68%ee chiralBuli,-n1)Ph Brn2)12 PhN2 FIG.37A FIG.37B atent Feb . 19 , 2019 Sheet 38 of 46 US 10 , 208 , 322 B2

*** NH2(Levomilnacipran)

X2=OH(8)or WX * *

Esterreductionor CNhydrogenation Ester!NOCYEt 2 ClopropanationCyclopropanation

y

* * ** * * denn Continente en X1=CO2Et,CN CONE2 U . S . Patent Feb . 19 , 2019 Sheet 39 of 46 US 10 ,208 , 322 B2

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X GKDPE MethodExpect BOX OM Ho QOWM *422QQFALSPATLVICMLXXFD403Sbjet 2893 ta 4 Graphics42211510Range1: GYLNFL310Query At Score . Query Query sbjct US 10 , 208 , 322 B2 IN VIVO AND IN VITRO OLEFIN highly substituted carbon centers and cyclopropanes , respec CYCLOPROPANATION CATALYZED BY tively ( H . M . L . Davies et al. , Chemical Reviews 103 , 2861 HEME ENZYMES (2003 ) ) . However, the most successful catalysts to date often utilize expensive and possibly toxic transition metal com CROSS -REFERENCES TO RELATED 5 plexes , with dirhodium species marking representative APPLICATIONS examples . Notably, high yield , regioselectivity , and stereo selectivity in these systems remains difficult to achieve and This application is a continuation of U .S . application Ser . many of these catalysts are hampered by harsh reaction No . 14 /625 ,449 , filed Feb . 18 , 2015 , now allowed , which conditions including high temperature and organic solvents . application is a continuation of U . S . application Ser. No . 10 The asymmetric cyclopropanation of olefins with high 14 / 185 , 861, filed Feb . 20 , 2014 , now issued as U . S . Pat . No . energy carbene precursors is a hallmark reaction that gen 8 , 993 , 262 on Mar. 31 , 2015 , which application is a continu erates up to 3 stereogenic centers in a single step to make the ation of PCT/ US2013 / 063577 , filed Oct . 4 , 2013 , which important cyclopropane motif , featured in many natural application claims priority to U .S . Provisional Application 15 products and therapeutic agents ( H . Lebel et al. , Chemical No . 61 / 711 ,640 , filed Oct . 9 , 2012 , U . S . Provisional Appli - 15 Reviews 103 , 977 ( 2003 ) ) . Limited to using physiologically cation No. 61/ 740 , 247 , filed Dec . 20 , 2012 , U . S . Provisional accessible reagents , Nature catalyzes intermolecular cyclo Application No. 61/ 784 ,917 , filed Mar . 14 , 2013 , U . S . propane formation through wholly different strategies, typi Provisional Application No. 61/ 838 , 167 , filed Jun . 21, 2013 , cally involving olefin addition to the methyl cation of U . S . Provisional Application No . 61/ 815 , 997 , filed Apr. 25 , 20 S - adenosyl methionine or through cyclization of dimethyl 2013 , U .S . Provisional Application No . 61/ 818 ,329 , filed 20 allyl pyrophosphate - derived allylic carbenium ions ( L . A . May 1 , 2013 , and U . S . Provisional Application No. 61 /856 , Wessjohann et al. , Chemical Reviews 103 , 1625 ( 2003 ) ) . As 493 , filed Jul. 19, 2013 . The disclosures of each of these a result, the diverse cyclopropanation products that can be applications and International Application No. PCT / formed by metallocarbene chemistry cannot be readily US2013 /63428 , filed Oct . 4 , 2013 , are hereby incorporated 25 accessed by engineering natural cyclopropanation enzymes. by reference in their entirety for all purposes. As such , there is a need in the art for novel reagents and catalytic schemes that are capable of creating the cyclopro STATEMENT AS TO RIGHTS TO INVENTIONS pane motif with high yield , regioselectivity , and stereose MADE UNDER FEDERALLY SPONSORED lectivity , but without the toxicity and harsh reaction condi RESEARCH AND DEVELOPMENT 30 tions associated with current approaches. The present This invention was made with government support under invention satisfies this need by providing novel iron -heme Grant No. DE -FG02 -06ER15762 / T - 20 105789 and Grant containing enzyme catalysts for producing cyclopropanation No. DE - FG02 -06ER15762 / T - 106029 awarded by the productspro in vitro and in vivo , and offers related advantages Department of Energy and under 1F32EB015846 -01 and as well. with government support under Grant No . EB015846 35 awarded by the National Institutes of Health . The govern SUMMARY OF THE INVENTION ment has certain rights in the invention . The present invention provides methods for catalyzing the REFERENCE TO A “ SEQUENCE LISTING ,” A conversion of an olefin to any compound ( e. g. , any inter TABLE . OR A COMPUTER PROGRAM LISTING 40 mediate or final compound ) containing one or more cyclo propane functional groups using heme enzymes . APPENDIX SUBMITTED AS AN ASCII TEXT In certain aspects , the present invention provides a FILE method for producing a cyclopropanation product, the The Sequence Listing written in file 086544 - 1022305 Se method comprising : quence Listing. txt, created on May 12 . 2017 . 417 .007 bytes . 45 ( a ) providing an olefinic substrate , a diazo reagent, and a heme enzyme ; and machine format IBM - PC ,MS -Windows operating system , is (b ) admixing the components of step ( a ) in a reaction for hereby incorporated by reference in its entirety for all a time sufficient to produce a cyclopropanation product. purposes . In some embodiments , the cyclopropanation product is a BACKGROUND OF THE INVENTION 50 compound according to Formula I: Aconsiderable challenge in modern synthetic chemistry is the selective direct functionalization of unactivated carbon hydrogen ( C - H ) bonds and carbon -carbon ( C = C ) double bonds (e .g . , olefins ) (R . G . Bergman , Nature 446 , 391 55 Šmps ( 2007 ) ; H . Pellissier, Tetrahedron 64 , 7041 ( 2008 ) ) . Adapt ing asymmetric catalytic processes to these reactions has 2 important consequences in the stereoselective and regiose RG lective elaboration of molecules for natural product and pharmaceutical synthesis . In recent years , much success has 60 wherein : been achieved in the development of catalysts for the select R ' is independently selected from the group consisting of addition of oxygen into molecules ( M . S . Chen et al ., H , optionally substituted C1- 18 alkyl, optionally substi Science 318 , 783 (2007 ) ) . More challenging is the direct tuted C6- 10 aryl , optionally substituted 6 - to 10 -mem introduction of new carbon - carbon centers into complex bered heteroaryl, halo , cyano , C ( O )ORIA , C ( O ) N (R ) 2 , structures . A contemporary catalytic approach uses metal- 65 C ( O ) R , C ( 0 ) C ( O )OR , and Si( Rº ) z ; locarbenoid intermediates that transfer a reactive carbene R2 is independently selected from the group consisting of into selectC H and C = C bonds, creating new asymmetric H , optionally substituted C1- 18 alkyl, optionally substi US 10 , 208 , 322 B2 tuted C6- 10 aryl, optionally substituted 6 - to 10 -mem bered heteroaryl, halo , cyano , C (O )OR24 , C ( O ) N (R7 ) 2, C ( O )R® , C ( O ) C ( O ) OR®, and Si ( R $ ) 3 ; wherein : Rla and R2a are independently selected from the group 5 Orla LORla, consisting of H , optionally substituted C1- 18 alkyl and - L - RC, wherein each L is selected from the group consisting of a bond , - C ( R ' ) 2 - , and - NR C ( R ) 2 - , each R is independently selected from the group LORla, consisting of H , C1- 6 alkyl, halo , CN , and — SO2, ma and each Rºis selected from the group consisting ofoption ally substituted C6- 10 aryl, optionally substituted 6 - 15 to 10 -membered heteroraryl, and optionally substi tuted 6 - to 10 -membered heterocyclyl; and LORla LORla R , R4, R ” , and R are independently selected from the ma na group consisting of H , C1- 18 alkyl, C2- 18 alkenyl , C2- 18 alkynyl, optionally substituted C6- 10 aryl, optionally 20 substituted C , -C6 alkoxy , halo , hydroxy , cyano , C ( O ) N ( R ') 2 , NR ' C ( O )RS , C (O )R® , C ( O )OR® , and N (Rº ) 2, wherein : each R7 and R8 is independently selected from the group consisting of H , optionally substituted C1- 12 25 LOR la OR la alkyl , optionally substituted C2- 12 alkenyl, and optionally substituted C6- 10 aryl; and each R is independently selected from the group con sisting of H , optionally substituted Co- 10 aryl, and optionally substituted 6 - to 10 -membered heteroaryl, 30 or two Rº moieties, together with the nitrogen atom to which they are attached , can form 6 - to 18 -mem OR la bered heterocyclyl; or R forms an optionally substituted 3 - to 18 -membered 25 ring with R4 ; or R $ forms an optionally substituted 3 - to 18 -membered ring with R ; ORIQ - ORla or R3 or R4 forms a double bond with R or R " ; or R3 or R4 forms an optionally substituted 5 - to 6 -mem - 40 bered ring with RS or R . In certain embodiments , R is C (O ) O -LR "; R2 is selected from the group consisting of H and optionally substituted C6- 10 aryl; and R *, R4 , R " , and Rºare independently selected Meo from the group consisting of H , optionally substituted C1- 6 45 LORla , 012 ORla alkyl, optionally substituted C2- 6 alkenyl, optionally substi NOM tuted C2- 6 alkynyl, optionally substituted C6- 10 aryland halo , or R $ forms an optionally substituted 3 - to 18 -membered ring with R * ; or Rºforms an optionally substituted 3 - to 50 18 -membered ring with Rº . OR la Orla In certain other embodiments , the cyclopropanation prod VA uct is a compound according to Formula II:

(II ) 55

R ? ( 0 - rla LORla

Rundvu bars, HO R4 wherein Rla is C1- 6 alkyl and R2 is selected from the group LORla consisting of H and optionally substituted C6- 10 aryl. 65 In some instances , R2 is H . In other instances , the cyclo propanation product is selected from the group consisting of: US 10 , 208 , 322 B2

- continued - continued

OR la COORla , wherein : Xl is selected from the group consisting of H , optionally 20 substituted C1- 6 alkyl , haloC1- 6 alkyl, optionally sub 10 stituted C1- 6 alkoxy, optionally substituted C1- 6 alkyl ORla - ORla thio , C1- 6 alkylsilyl, halo , and cyano ; X² is selected from the group consisting of H , chloro , and methyl; X® is selected from the group consisting of H , methyl , Ci Ci 15 halo , and CN ; each X4 is independently halo ; each XS is independently selected from the group con ' COORla , mann LORla sisting of methyl and halo , X® is selected from the group consisting of halo , option 20 ally substituted Ci- alkyl, and optionally substituted C1- 6 alkoxy ; X7 is selected from the group consisting of H , methyl, and halo ; X® is selected from the group consisting of H , halo , and LORla , 25 optionally substituted C . , alkyl; X® is selected from the group consisting of H , halo , optionally substituted C1- 6 alkyl , C ( O ) O - (C1 - 6 alkyl) , yl C ( O ) - N ( C1- 6 alkyl) 2 , and cyano ; and Z ' , Z² , and Z are independently selected from the group 30 consisting of H , halo , optionally substituted C1- 6 alkyl , Orla Orla and optionally substituted C6- 10 aryl; or ma z and Z ? are taken together to form an optionally sub stituted 5 - to 6 -membered cycloalkyl or heterocycly1 group . In certain embodiments , the method further comprises 35 converting the cyclopropanation product to a compound according to Formula III:

Orla , to COORla , 40 R? 40 - LRIC Ring Ames, 45 DR4 COORla , wherein : L is selected from the group consisting of a bond , 50 - C ( R ) 2 - , and — NR C ( R ) 2 - , each R is independently selected from the group con sisting of H , — CN , and — SO2, and Rlc is selected from the group consisting of optionally subsisted C6- 10 aryl, optionally substituted 6 - to COORla, 55 10 -membered heteroraryl, and optionally substituted 6 to 10 -membered heterocyclyl. In some instances, L is selected from the group consisting of a bond , CH2 - , - CH ( CN ) — , and — N (SO2 ) CH . In other instances , the moiety L -Rle has a structure selected LÅ COORla , z2 LX la 60 from the group consisting of: COORla , and . .65 01 US 10 ,208 , 322 B2

- continued -continued

??????? 10 ?? 15 ??? ???????? 20 | ?????? 25 ???? 30 - ????????? 35

40)

.1

45

?????? 50

55

09( ( ? ?) ?,

65 US 10 , 208 , 322 B2 10 - continued In some instances , the cyclopropanation product has a structure selected from the group consisting of:

A (CH2 ) COOE ETOOC Et2NOCH JOULICONET? PLPh H3 10 y5, and Eto - NH2

CH3, cordoglio& # 1131 Eto ORS

20 wherein : each Yl is independently selected from the group con sisting of optionally substituted C1- 6 alkyl, optionally 25 substituted C2- 6 alkenyl, optionally substituted C2- 6 alkynyl, phenyl, and (phenyl ) C1 - 6 alkoxy ; each Y2 is independently selected from the group con Cl, and sisting ofhalo , optionally substituted C1- 6 alkyl, option - 30 ally substituted C2- 6 alkenyl , optionally substituted C1- 6 alkoxy, and nitro ; Eto NH , each Yº is independently optionally substituted C - 6 alkyl; each Y4 is independently selected from the group con sisting of optionally substituted C1- 6 alkyl, optionally 35 substituted C2- 6 alkenyl, optionally substituted C2 -6 alkynyl, C6 -10 aryl- C1 - 6 alkyl, furfuryl , C1- 6 alkoxy , (C2 - 6 alkenyl) oxy , C1- 12 acyl, and halo ; Y is selected from the group consisting of optionally Eto - ORS substituted C1- 6 alkyl, optionally substituted C1- 6 40 alkoxy, and halo , the subscript m is an integer from 1 to 3 , In certain instances, the method further comprises con the subscript n is an integer from 1 to 5 , verting the cyclopropanation product to a compound the subscript p is an integer from 1 to 4 , selected from the group consisting of milnacipran , levom the subscript q is an integer from 0 to 3 , ilnacipran , bicifadine, and 1 - (3 , 4 -dichlorophenyl ) - 3 - azabi and the wavy line at the left of the structure represents the cyclo [ 3 . 1 . 0 ]hexane . point of connection between the moiety - L -RIC and the In some embodiments, the olefinic substrate is selected rest of the compound according to Formula III . from the group consisting of an alkene , a cycloalkene , and In yet other instances, the compound according to For- an arylalkene . In certain instances, the olefinic substrate mula III is resmethrin . 50 comprises an arylalkene . In some instances , the arylalkene is In certain embodiments, the cyclopropanation product is a styrene . In other instances , the styrene has the formula : a compound having a structure according to the formula :

55

0 - Rla , (R19 , PAN R6 Pom 60 wherein R ’ is selected from the group consisting of H , wherein : optionally substituted C1- C , alkyl, optionally substi Rla is optionally substituted C1- 6 alkyl, and tuted C , -C alkoxy, C ( O ) N ( R )2 , C (O )OR® , N (R )2 , R and R are independently selected from the group halo , hydroxy, and cyano ; consisting of H , optionally substituted C1- 6 alkyl, 65 R and Rº are independently selected from the group optionally substituted C6- 10 aryl, C (O ) N (R ) 2, C ( O ) consisting of H , optionally substituted C1- 6 alkyl, and OR® and NR ? C (O ) R8. halo ; US 10 , 208 , 322 B2 11 12 Riº is selected from the group consisting of optionally alkyl, optionally substituted C2- 12 alkenyl , and substituted C , -Co alkyl, optionally substituted C1- C6 optionally substituted C6- 10 aryl. alkoxy , halo , and haloalkyl ; and In certain other instances, the diazo reagent is selected the subscript r is an integer from 0 to 2 . from the group consisting of diazomethane , ethyl diazoac In some embodiments , the diazo reagent has a structure 5 etate , and ( trimethylsilyl )diazomethane . In yet other according to the formula : instances , the diazo reagent has the formula :

R ? R 10 Rio wherein : R is independently selected from the group consisting of 15 H , optionally substituted C1- 18 alkyl, optionally substi In particular embodiments , the cyclopropanation product tuted C6- 10 aryl , optionally substituted 6 - to 10 -mem has a formula selected from the group consisting of: bered heteroaryl, halo , cyano , C ( O )ORIA , C ( O ) N (R )2 , C ( O ) RS, C ( O ) C ( O ) ORS, and Si( R )z ; and R is independently selected from the group consisting of - RS R6 H , optionally substituted C1- 18 alkyl, optionally substi Spo tuted C6- 10 aryl, optionally substituted 6 - to 10 -mem COORla bered heteroaryl, halo , cyano , C ( O )OR2a , C ( O ) N ( R ? )2 , and C (O )R® , C ( O ) C ( O ) OR®, and Si (R $ )3 ; 25 RS RG wherein Eas Rla and R24 are independently selected from the group consisting of H , optionally substituted C1- 18 alkyl A COOR and - L - R “ , wherein each L is selected from the group consisting of a bond , 30 C (R )2 - , and NR - C ( R ) 2 - , (R10 each R is independently selected from the group consisting of H , C1- 6 alkyl, halo , CN , and SO2, and In some embodiments , the method is carried out in vitro . each R is selected from the group consisting of option - 35 In certain instances , the reaction further comprises a reduc ally substituted Ca 1 aryl, optionally substituted 6 - ing agent. In other embodiments, the heme enzyme is to 10 -membered heteroraryl, and optionally substi localized within a whole cell and the method is carried out in vivo . In some embodiments , the method is carried out tuted 6 - to 10 -membered heterocyclyl; and under anaerobic conditions. each R7 and R8 is independently selected from the group 40 In some embodiments , the method produces a plurality of consisting of H , optionally substituted C1- 12 alkyl, cyclopropanation products . In certain instances, the plurality optionally substituted C2- 12 alkenyl , and optionally of cyclopropanation products has a Z : E ratio of from 1 : 99 to substituted C6- 10 aryl. 99 : 1 . In some instances , the plurality of cyclopropanation In certain embodiments , the diazo reagent is selected from products has a % eez of from about - 90 % to about 90 % . In the group consisting of an a -diazoester , an a -diazoamide , an 45 other instances, the plurality of cyclopropanation products a - diazonitrile , an d - diazoketone , an a - diazoaldehyde , and has a % eer of from about - 90 % to about 90 % . In some an a - diazosilane . instances, the cyclopropanation reaction is at least 30 % to at In certain instances , the diazo reagent has a formula least 90 % diasteroselective . In other instances , the cyclo selected from the group consisting of: propanation reaction is at least 30 % to at least 90 % enanti 50 oselective . In some embodiments , the heme enzyme is expressed in a bacterial, archaeal, or fungal host organism . Rio In certain embodiments, the heme enzyme is a fragment ) N (R ?) 2 thereof comprising the heme domain . In particular embodi ments , the heme enzyme is an engineered heme enzyme doloN2 N2N2 such as a heme enzyme variant comprising a mutation at the O O axial position of the heme coordination site . In some Si( R $) 3 instances , the mutation is a substitution of the native residue 8 R8K ,? and a with Ala , Asp , Arg , Asn , Cys , Glu , Gln , Gly , His , Ile , Lys , 60 Leu , Met , Phe , Pro , Ser, Thr, Trp , Tyr , or Val at the axial position . In certain instances, the mutation is a substitution of Cys with Ser at the axial position . In other embodiments , wherein the heme enzyme variant is a chimeric protein comprising Rla is selected from the group consisting of H and recombined sequences or blocks of amino acids from two , Where is selected from theco -C . alkyl; ander from the 65 three , or more different heme- containing proteins . each R7 and R8 is independently selected from the In particular embodiments , the heme enzyme is a cyc group consisting of H , optionally substituted C1- 12 tochrome P450 enzyme or a variant thereof. In preferred US 10 , 208 , 322 B2 13 14 embodiments , the cyctochrome P450 enzyme is a P450 substitution of Cys with Ala , Asp , Arg , Asn , Glu , Gin , Gly , BM3 enzyme or a variant thereof. His , Ile , Lys , Leu , Met, Phe , Pro , Ser , Thr , Trp , Tyr, or Val In certain embodiments , the cyctochrome P450 enzyme at the axial position . In certain instances, the mutation is a variant comprises a mutation at the axial position of the substitution of Cys with Ser at the axial position . In other heme coordination site . In some instances , the mutation is a 5 embodiments , the cyctochrome P450 variant is a chimeric substitution of Cys with Ala , Asp , Arg , Asn , Glu , Gin , Gly , protein comprising recombined sequences or blocks of His , Ile , Lys , Leu , Met, Phe, Pro , Ser, Thr, Trp , Tyr, or Val amino acids from at least two , three , or more different P450 at the axial position . In certain instances, the mutation is a enzymes ( e . g. , CYP102A1, CYP102A2, and CYP102A3 ) . substitution of Cys with Ser at the axial position . In other In certain embodiments , the P450 BM3 enzyme variant embodiments, the cyctochrome P450 variant is a chimeric 10 comprises at least one mutation in the amino acid sequence protein comprising recombined sequences or blocks of set forth in SEQ ID NO : 1 . In particular embodiments , the amino acids from at least two , three , or more different P450 P450 BM3 enzyme variant comprises a C400X mutation at enzymes ( e . g . , CYP102A1 (P450 BM3) , CYP102A2 , and the axial position in SEQ ID NO : 1 , wherein X is any amino CYP102A3 ) . acid other than Cys . In other embodiments , the P450 BM3 In some embodiments , the P450 BM3 enzyme comprises 15 enzyme variant comprises at least one , two , three , four, five , the amino acid sequence set forth in SEQ ID NO : 1 . In six , seven , eight, nine, ten , eleven , twelve , or all thirteen of particular embodiments , the P450 BM3 enzyme variant the following amino acid substitutions in SEQ ID NO : 1 : comprises a C400X mutation at the axial position in SEQ ID V78A , F87V , P142S , T1751, A184V , S226R , H236Q , NO : 1 , wherein X is any amino acid other than Cys. In other E252G , T268A , A290V, L353V , 1366V , and E442K . embodiments , the P450 BM3 enzyme variant comprises at 20 In some embodiments, the P450 BM3 enzyme variant least one , two, three , four, five, six , seven , eight, nine , ten , comprises at least one, two , or all three of the following eleven , twelve , or all thirteen of the following amino acid amino acid substitutions in SEQ ID NO : 1 : 1263A , A328G , substitutions in SEO ID NO : 1 : V78A , F87V , P142S , T1751, and a T438 mutation . In certain instances , the T438 mutation A184V , S226R , H236Q , E252G , T268A , A290V , L353V , is T438A , T438S , or T438P . 1366V , and E442K . 25 In some embodiments , the P450 BM3 enzyme variant In some embodiments , the P450 BM3 enzyme variant comprises from one to five alanine substitutions in the active comprises at least one , two, or all three of the following site of SEQ ID NO : 1 . In certain instances , the active site amino acid substitutions in SEQ ID NO : 1: 1263A , A328G , alanine substitutions are selected from the group consisting and a T438 mutation . In certain instances, the T438 mutation of L75A , M177A , L181A , 1263A , L437A , and a combina is T438A , T438S , or T438P . 30 tion thereof. In some embodiments, the P450 BM3 enzyme variant In particular embodiments, the P450 BM3 enzyme variant comprises from one to five alanine substitutions in the active comprises a T268A mutation and / or a C400X mutation in site of SEQ ID NO : 1 . In certain instances , the active site SEQ ID NO : 1 , wherein X is any amino acid other than Cys . alanine substitutions are selected from the group consisting I n some embodiments , the heme enzyme variant fragment of L75A , M177A , L181A , 1263A , L437A , and a combina - 35 comprises the heme domain thereof. In particular embodi tion thereof. ments , the heme enzyme variant is a P450 enzyme variant In particular embodiments , the P450 BM3 enzyme variant selected from Tables 4 , 5A , and 5B . comprises a T268A mutation and / or a C400X mutation in In other embodiments , the heme enzyme variant has a SEQ ID NO : 1 , wherein X is any amino acid other than Cys. higher total turnover number ( TTN ) compared to the wild In some embodiments , the heme enzyme comprises a 40 type sequence . In certain instances , the heme enzyme variant fragment of the cytochrome P450 enzyme or variant thereof. has a TTN greater than about 100 . In certain instances, the fragment comprises the heme In some instances, the heme enzyme variant produces a domain of the cyctochrome P450 enzyme or variant thereof. plurality of cyclopropanation products having a Z : E ratio of In particular embodiments, the heme enzyme is a P450 from 1 : 99 to 99 : 1 . In some instances, the heme enzyme enzyme variant selected from Tables 4 , 5A , and 5B . 45 variant produces a plurality of cyclopropanation products In other aspects , the present invention provides a heme having a % ee , of at least - 90 % to at least 90 % . In other enzyme or a fragment thereof that can cyclopropanate an instances , the heme enzyme variant produces a plurality of olefinic substrate . cycyclopropanation products having a % eer of at least - 90 % In particular embodiments , the heme enzyme is an engi- to at least 90 % . In some instances , the heme enzyme variant neered heme enzyme such as a heme enzyme variant com - 50 produces a plurality of cyclopropanation products having at prising a mutation at the axial position of the heme coordi- least 30 % to at least 90 % diasteroselectivity . In other nation site . In some instances , the mutation is a substitution instances , the heme enzyme variant produces a plurality of of the native residue with Ala , Asp , Arg , Asn , Cys , Glu , Gin , cyclopropanation products having at least 30 % to at least Gly, His, Ile , Lys, Leu , Met , Phe , Pro , Ser , Thr , Trp , Tyr, or 90 % enantioselectivity . In yet other instances , the heme Val at the axial position . In certain instances, the mutation is 55 enzyme variant is in lyophilized form . a substitution of Cys with Ser at the axial position . In other In further aspects, the present invention provides a cell embodiments , the heme enzyme variant is a chimeric protein expressing a heme enzyme described herein . In certain comprising recombined sequences or blocks of amino acids embodiments , the cell is a bacterial cell or a yeast cell . from two , three, or more different heme- containing proteins. In yet other aspects , the present invention provides an In some embodiments, the heme enzyme variant is iso - 60 expression vector comprising a nucleic acid sequence lated and /or purified . In other embodiments , the heme encoding a heme enzyme described herein . In related enzyme variant is a cyctochrome P450 enzyme variant. In aspects , the present invention provides a cell comprising the preferred embodiments , the cyctochrome P450 enzyme vari expression vector. In certain embodiments , the cell is a ant is a P450 BM3 enzyme variant . bacterial cell or a yeast cell . In certain embodiments , the cyctochrome P450 enzyme 65 Other objects , features, and advantages of the present variant comprises a mutation at the axial position of the invention will be apparent to one of skill in the art from the heme coordination site . In some instances , the mutation is a following detailed description and figures. US 10 , 208 , 322 B2 15 16 BRIEF DESCRIPTION OF THE DRAWINGS changes were observed (RMSD 0 .52 Å ). Middle panels : Large variations are observed upon comparing P411BM3 -CIS FIG . 1 illustrates the canonical mode of reactivity of with the open ( ligand - free ) form of wild type P450BM3 cytochrome P450s . (Left ): monooxygenation of olefins and (purple , taken from PDB # 21J2 , RMSD 1 .2 Å ) . Pronounced C - H bonds to epoxides and alcohols catalyzed by the ferryl 5 rearrangements are observed in active site side chain resi porphyrin radical intermediate ( Compound I ) . ( Right ): Arti ficial mode of formal carbene transfer activity of dues ( left) as well as rotations within the I -helix . Global cytochrome P450s utilizing diazoester reagents as carbene movements are also observed in the N - terminal beta domain precursors. as well as F - and G -helices (right ,marked by double headed FIG . 2 illustrates the absolute stereoselectivity of select 10 arrows) . These movements are consistent with well - known P450 Bm cyclopropanation catalysts . Reaction conditions : transitions that occur upon substrate binding and are impor 20 uM catalyst , 30 mM styrene , 10 mM EDA , 10 mm tant for native monooxygenation catalysis . Bottom panels : Na, s , o , , under argon in aqueous potassium phosphate Alignment of P450BM3 - CIS with a ligand -bound P450BM3 buffer (pH 8 . 0 ) and 5 % MeOH cosolvent for 2 hours at 298 structure (cyan , taken from PDB # 1. JPZ , RMSD 0 . 52 Å ) K . Enzyme loading is 0 . 2 mol % with respect to EDA . The 15 demonstrates that P450BM3- CIS and P4113M3- CIS mimic structures of each product stereoisomer are shown above the the closed protein conformation even in the absence of reaction gas chromatograms. substrate . Protein alignments were carried out using the FIG . 3 illustrates the effect of styrene concentration on align tool of PyMol (PyMOL Molecular Graphics System , cyclopropane yield . Version 1 . 3 Schrödinger , LLC . ). FIG . 4 illustrates the effect of P450 (H2A10 ) concentra - 20 FIG . 10 illustrates that whole -cell cyclopropanation cata tion on cyclopropane yield . lysts are inhibited under aerobic conditions. Variant 9 - 10A FIGS . 5A - B illustrate the initial velocities plot for variant TS -F87V - T268A = BM3- CIS and ABC = BM3 + C400S . Reac 9 - 10A - TS - F87V - T268A (C3C ) heme: ( FIG . 5A ) EDA concen - tion conditions: 20 mM styrene , 10 mM EDA , under argon tration was varied at a saturating concentration of styrene (or air ) in nitrogen - free medium and 5 % MeOH cosolvent ( 30 mM ) . ( FIG . 5B ) Styrene concentration was varied at a 25 for 2 hours at 298 K (OD . O . 24 ) . Total fixed concentration of EDA ( 20 mM ) . Initial rates were turnover = concentration of cyclopropanes (mM ) /cell density computed as the slope of a zero - intercept linear fit of three ( g cdw / L ) in units of mmol/ g cdw . different time points from independent reactions. Error bars FIG . 11 illustrates the effect of glucose addition on in vivo correspond to 1 - 0 (68 . 3 % ) confidence intervals for the cyclopropanation of styrene. Reaction conditions : 20 mM slope . 30 FIG . 6 illustrates the cyclopropanation activities of vari styrene, 10 mM EDA , under argon in nitrogen - free medium ant 9 - 10A - TS - F87V - T268A (also called BM3- CIS or and 5 % MeOH cosolvent for 2 hours at 298 K (OD600 ~ 24 ) . P4508M3 -CIS ) and BM3- CIS - C400S ( also called ABC -CIS Total turnover = concentration of cyclopropanes (mM ) / cell or P4113M3 - CIS ) driven by sodium dithionite under anaero density ( g cdw / L ) in units of mmol/ g cdw . Variant 9 - 10A bic and aerobic conditions. Measurements were taken in 35 TS -F87V - T268A = BM3- CIS and ABC = BM3 + C400S . triplicate and the error bars represent the standard deviation FIG . 12 illustrates the effect of media and comparison of from the mean value . holo vs . heme forms of BM3- CIS -C400S on in vivo cyclo FIG . 7 illustrates the cyclopropanation and epoxidation propanation of styrene . Reaction conditions: 20 mM styrene , activities of variant 9 - 10A - TS - F87V - T268A ( also called 10 mM EDA , 2 mM glucose under argon in nitrogen - free BM3- CIS or P450 -CIS ) and BM3- CIS -C400S ( also 40 medium and 5 % MeOH cosolvent for 2 hours at 298 K called ABC -CIS or P411 BM3- CIS ) driven by NADPH under (OD600 ~ 24 ) . Variant 9 - 10A - TS - F87V - T268A = BM3- CIS anaerobic and aerobic conditions . Measurements were taken and ABC = BM3 + C400S . in triplicate and the error bars represent the standard devia - FIG . 13 illustrates that increasing ABC catalyst loading tion from the mean value . Reaction conditions : 20 uM (cell density ) increases cyclopropanes yield up to approxi catalyst , 30 mM styrene , 10 mM EDA , 0 . 5 mM NADPH , 25 45 mately 80 % at OD .. . = 50 . ABC (BM3 - C400S or P411 RM ) . mM glucose , 2 U ml- 2 glucose dehydrogenase under argon FIG . 14 illustrates the effect of using 1 , 2 , 3 , 4 and 5 ( or air ) in aqueous potassium phosphate buffer (pH 8 .0 ) and equivalents of styrene on reaction yield . Excess styrene 5 % MeOH cosolvent for 2 hours at 298 K . Measurements gives only small improvements in yield .Measurements were were taken in triplicate and the error bars represent the taken in triplicate and the error bars represent the standard standard deviation of the measurements . FIGS. 8A - B illustrate the initial velocities plot for BM3 O deviation from the mean value . ABC (BM3 -C400S or CIS -C400S ( also called ABC -CIS or P411BM3- CIS ) heme. P411BM3) . ( FIG . 8A ) EDA concentration was varied at a saturating FIG . 15 illustrates controls for ABC catalyzed cyclopro concentration of styrene ( 30 mM ) . (FIG . 8B ) Styrene con panation . Variant 9 - 10A - TS -F87V - T268A = BM3- CIS and centration was varied at a fixed concentration of EDA (20 55 ABC = BM3- C400S . mM ) . Initial rates were computed as the slope of a zero FIG . 16 illustrates that ABC catalyst is active for 3 hours . intercept linear fit of three different time points from inde At OD600 = 25 , the P450 concentration in 0 .85 uM , such that pendent reactions. Error bars correspond to 1 - 0 (68 . 3 % ) TTN > 8 , 000 . Variant 9 - 10A - TS - F87V - T268A = BM3- CIS confidence intervals for the slope . and ABC = BM3- C400S . FIG . 9 illustrates the heme domain active site and protein 60 FIGS . 17A - D illustrate the proposed substrate scope of alignments of variant 9 - 10A - TS -F87V - T268A ( also called P450 -catalyzed cyclopropanation . ( FIG . 17A ) Diazo -com BM3- CIS or P450BM3 -CIS ) with BM3- CIS - C400S ( also pounds that can be used as the carbenoid precursor. (FIG . called ABC -CIS or P411BM3- CIS ) and wild type P4508M3. 17B ) Olefin partners for reaction . (FIG . 17C ) Cyclopropa Top panels shows alignments of P450RM3 -CIS ( green ) and nation of geranyl acetone for synthesis of anthroplalone and P411BM3 -CIS (peach ) with left, middle and right panels 65 noranthroplone. (FIG . 17D ) Cyclopropanation of 2 , 5 - dim showing active site residues, the active site I - helix , and ethylhexa - 2 , 4 - diene in the synthesis of pyrethroid insecti global protein fold , respectively . No significant structural cides . US 10 , 208 , 322 B2 18 FIG . 18 illustrates UV - Vis absorption spectra of purified metric addition of sodium dithionite (dashed line : fully

BM3- CISheme ( 3 uM , red line ) and BM3- CIS - C400Shemee ( 2 ferric ; solid line: fully ferrous) . UM , green line ) . Insert shows solutions of both proteins at FIG . 26 illustrates the potentiometric redox titration for approximately 1 mM . P450 RM3- home -CIS with overlaid Nernst curve fit to E 01 = FIGS. 19A - C illustrate ( FIG . 19A ) Cytochrome P450s 5 360 mV. Inset shows spectral changes upon each sub inefficiently catalyze cyclopropanation using NAD (P )H as a stoichiometric addition of sodium dithionite ( dashed line : fully ferric ; solid line: fully ferrous ) . reductant because the Fem /Fe " redox potential for the low FIG . 27 illustrates the potentiometric redox titration for spin resting state (E° ' Fe - cus = - 430 mV ) is lower than that of P411373 -heme -CIS with overlaid Nernst curve fit to E = NAD ( P ) + /NAD (P ) H ( E O ' = - 320 mV ) . Replacement of the 10 265 mV. Inset shows spectral changes upon each sub heme- cysteine ligand (Fe - Cys ) with serine (Fe -Ser ) stoichiometric addition of sodium dithionite (dashed line : increased the resting state reduction potential and allowed fully ferric ; solid line : fully ferrous) . reduction by NAD ( P ) H to the active Fet species in vivo . FIG . 28 illustrates the cyclopropanation activity under ( FIG . 19B ) Close -up of the ABC - CIS active site (PDB : anaerobic vs . aerobic conditions with dithionite in variant 4H24 ) superimposed with an F , - FF simulated annealing 15 9 - 10A - TS - F87V - T268A ( also called BM3 - CIS or omit map contoured at 30 showing electronelectron density ( green P450 BM3- heme- CIS ) and BM3- CIS - C400S (also called ABC mesh ) corresponding to the bound heme and C400S muta - CIS or P411 -CIS ) . Measurements were taken in tion . Interconnected density between C400S and the heme triplicate and the error bars represent the standard deviation iron is consistent with proximal heme ligation by the C400S of the measurements . side chain hydroxyl. The heme, C400S and additional active 20 FIG . 29 illustrates the effect of adding exogenous glucose site amino acid side chains are shown as sticks . ( FIG . 19C ) ( 2 mM ) on olefin cyclopropanation catalyzed by E . coli Potentiometric redox titrations for BM3 ( green circles ) and whole cells expressing 9 - 10A - TS - F87V - T268A (also called ABC ( blue triangles) with overlaid one- electron Nernst BM3- CIS and P450BM3 -CIS ) or BM3- CIS - C400S ( also curves . Insets show spectral changes between ferric (dashed called ABC - CIS and P411BMS - CIS ). line ) and ferrous ( solid line ) states. The changes in absor - 25 FIG . 30 illustrates the thermostabilities of heme domains bance near 450 - 470 nm (BM3 ) and 420 -440 ( ABC ) were of BM3- CIS (P450BM3 - CIS in blue ) and ABC - CIS used to determine the Fe' / Fel ratio after reduction with ( P4111 -CIS in red ) . The C400S mutation stabilizes the dithionite . The reduction is reversible , and reoxidation by heme domain by + 1 . 7° C . The Tso is the temperature at potassium ferricyanide shows little or no hysteresis . The which half of the enzyme population has unfolded . Error midpoint potential of the serine - ligated mutant ( - 293 mV) is 30 bars correspond to 1 - 0 (68 . 3 % ) confidence intervals for the shifted 127 mV positive compared to WT ( - 420 mV ) . T50 . FIGS . 20A - B illustrate the heme electron density of FIG . 31 illustrates the effect of dioxygen exposure on BM3- CIS -C400S (also called ABC -CIS or P411BM3 - CIS ). whole- cell catalyzed cyclopropanation . ABC -CIS Maximum likelihood weighted electron density maps of (P411 BM3 - CIS ) is strongly inhibited by dioxygen in vivo . All serine - ligated heme in P411BM3 - CIS . (FIG . 20A ) Stereo 35 reactions had a cell density equivalent to OD600 = 25 . Reac image of heme bound to P411 BM3 -CIS viewed from the top tions were conducted in the absence of exogenous glucose . of the heme in the active site . (FIG . 20B ) Stereo image of Measurements were taken in triplicate and the error bars heme bound to P411BM3 - CIS rotated - 90° from panel A represent the standard deviation from the mean value. Prod shows clear indication of heme- iron ligation by the side uct formation is defined as the amount of cyclopropane chain hydroxyl of C400S . All atoms are shown as sticks . All 40 product (mmol ) formed per mass of catalyst ( g ) . electron density maps were contoured at o = 1. 0 . For per - FIG . 32 illustrates the empty plasmid , no - induction con spective, in panel B , the main chain atoms of residues 399 t rols and dithionite addition to whole cells . E . coli cells and 401 are also shown as sticks . carrying the ABC - CIS ( P4113M3 - CIS ) gene but grown with FIG . 21 illustrates the absolute spectra for ferric (blue ), out the addition of IPTG ( ABC - CIS no induction ) ; E . coli dithionite reduced ferrous (red ) and carbon monoxide bound 45 cells carrying the pc Wori plasmid but not the ABC -CIS gene ferrous ( green ) ABC -CISheme . Soret bands ( nm ): Fe , 404 ; ( empty pc Wori ); ABC -CIS reaction with the addition of Fe " , 410 , 425 ; Fett CO , 411. Fem CO displays a and ß exogenous dithionite instead of glucose (ABC - CIS + dithi bands at 532 and 565 nm . Insert shows the carbon monoxide onite ). Reactions were left for two hours at 298 K . Mea ferrous (pink ) and the dithionite reduced ferrous (yellow ) surements were taken in triplicate and the error bars repre enzymes at 4 . 5 uM protein concentration . 50 sent the standard deviation from the mean value . Product FIG . 22 illustrates the absolute spectra for ferric (blue ), formation is defined as the amount of cyclopropane product dithionite reduced ferrous ( red ) and carbon monoxide bound (mmol ) formed per mass of catalyst ( Scdw ). ferrous ( green ) ABC - CIShoe. Soret bands (nm ): Femm , 404 ; FIG . 33 illustrates that increasing cell density increases Fe " , 410 , 422 ; Fer _ CO , 411 . Fer - CO displays a and B cyclopropane yields up - 80 % . Total turnovers do not bands at 533 and 566 nm . Ferric spectrum displays a broad 55 increase for cell densities higher than OD . 00 = 20 . Measure peak at 465 nm . ments were taken in triplicate and the error bars represent the FIGS . 23A - B illustrate the difference spectra for ferrous standard deviation from the mean value . Product formation carbonyl with respect to ferrous for : (FIG . 23A ) ABC is defined as the amount of cyclopropane product ( mmol) CIS home and ( FIG . 23B ) ABC -CIS holo . formed per mass of catalyst ( gedu ) . FIG . 24 illustrates the potentiometric redox titration for 60 FIG . 34 illustrates the time- course for in vivo and in vitro P450 BM3- heme with overlaid Nernst curve fit to E " = -420 ABC - CIS (P411BM3 -CIS )- catalyzed reaction at P411 load mV . Inset shows spectral changes upon each sub -stoichio - ing of 1 .6 UM [8411 - 490 = 103 mM - cm for the ferrous -CO metric addition of sodium dithionite (dashed line : fully complex ( K . P . Vatsis et al. , J. Inorg . Biochem . 91, 542 ferric ; solid line : fully ferrous) . (2002 ) ) ] . Reaction conditions were as follows: 40 mM FIG . 25 illustrates the potentiometric redox titration for 65 styrene , 20 mM EDA, 2 mM glucose , 10 . 2 gedw L - 1 whole P411 BM3- heme with overlaid Nernst curve fit to E " = - 293 cell ABC -CIS (in vivo ), 1 .6 uM purified ABC - CIS ( in vitro ) mV. Inset shows spectral changes upon each sub - stoichio - in aqueous nitrogen - free M9 minimal medium and 5 % US 10 , 208 , 322 B2 10 20 MeOH cosolvent under anaerobic conditions at 298 K . FIG . 45A shows the sequence alignment between Yields at each time point are reported as averages of two cytochrome P450 BM3 (SEQ ID NO :76 ) and CYP2D7 independent reactions. ( SEQ ID NO :75 ). The C400 axial ligand in P450 BM3 FIGS . 35A - C illustrate the contrasting P450 - and P411 - corresponds to C461 in CYP2D7. FIG . 45B shows the a cyclopropanation . (FIG . 35A ) Cytochrome P450s 5 sequence alignment between cytochrome P450 BM3 ( SEO inefficiently catalyze cyclopropanation using NAD ( P ) H as a ID NO :78 ) and P450C27 (SEO ID NO :77 ) . The C400 axial reductant because the Fe 7Fe redox potential for the low ligand in P450 BM3 corresponds to C478 in P450C27 . spin resting state ( E '' Fe- cus = - 430 mV ) is lower than that of NAD ( P ) + /NAD ( P ) H ( E 0 = - 320 mV, right) . Mutation of DETAILED DESCRIPTION OF THE the heme- ligating Cys to Ser allows NAD ( P ) H - driven cyclo - 10 INVENTION propanation while removing native monooxygenation (left ) . ( FIG . 35B ) Close - up of the P411BM3 -heme -CIS active site I. Introduction ( PDB : 4H24 ) superimposed with an F . -Fe simulated anneal ing omit map contoured at 30 showing electron density The present invention is based on the surprising discovery ( green mesh ) corresponding to the bound heme and C400S 15 that heme enzymes can be used to catalyze the conversion of mutation . Heme, C400S and additional active site amino olefins to any product containing one or more cyclopropane acid side chains are shown as sticks. (FIG . 35C ) In vitro functional groups. In some aspects , cytochrome P450 cyclopropanation vs . epoxidation of styrene catalyzed by enzymes ( e .g ., P450 BM3 (CYP102A1 ) ) and variants P450BM3 -CIS and P411BM3 -CIS under anaerobic and aero thereof were identified as having an unexpectedly improved bic conditions. Reaction conditions were as follows: 30 mM 20 ability to catalyze the formal transfer of carbene equivalents styrene , 10 mM EDA , 0 . 5 mM NADPH , 25 mM glucose , 2 from diazo reagents to various olefinic substrates , thereby U ml- glucose dehydrogenase and 20 g enzyme in aqueous making cyclopropane products with high regioselectivity potassium phosphate buffer and 5 % MeOH cosolvent for six and /or stereoselectivity . In particular embodiments , the pres hours at 25° C . Error bars represent the standard deviation ent inventors have discovered that variants of P450 By with of three independent measurements. 25 at least one or more amino acid mutations such as an axial FIGS . 36A - C illustrate the racemic synthesis ofmilnacip ligand C400X ( e . g ., C400S ) and / or an T268A amino acid ran . ( FIG . 36A ) Synthesis ofkey intermediate 1 . ( FIG . 36B ) substitution can catalyze cyclopropanation reactions effi Routes A - C for conversion of 1 to milnacipran . ( FIG . 36C ) ciently , displaying increased total turnover numbers (TTN ) Route to milnacipran based on selective deprotonation of 5 . and demonstrating highly regio - and enantioselective prod FIGS. 37A - B illustrate the enantioselective synthesis of 30 uct formation compared to wild - type enzymes. levomilnacipran using (FIG . 37A ) Rh -catalyzed intramo- As a non -limiting example , axial serine heme ligation lecular cyclopropanation or (FIG . 37B ) asymmetric alky - (C400S in BM3 ) in cytochrome P450s creates the homolo lation using tetraamine chiral auxiliary . gous " cytochrome P411 ” family , which catalyze the cyclo FIG . 38 illustrates the synthesis of levomilnacipran via propanation reaction in vivo in whole cells , providing over carbene cyclopropanation by cytochrome P450 BM3 variants. 35 10 , 000 total turnovers with high stereoselectivity , optical FIG . 39 illustrates the gas chromatography trace of reac - purity and yield , making the cyclopropane product with tion of 9b and EDA mediated by BM3- CIS -AxH , using titers of over 20 g L - 7 . Thus , the cytochrome P411 family is Agilent cycloSil - B column , 30 mx0 .25 umx0 . 32 mm , spectroscopically , electrochemically , and catalytically dis method : 90° C . (hold 2 min ) , 90 - 110 (6° C ./ min ) , 110 - 190 tinct from cytochrome P450s , providing a scaffold for engi ( 40° C ./ min ), and 190 -280 (20° C ./ min ). Internal standard at 40 neering orthogonal heme- enzyme catalysis . As such , the 4 . 8 min , starting material at 10 . 4 min , and product at 14 . 8 ability to catalyze this non - natural C - Cbond forming min ( E - isomer ) and 15 . 4 min ( Z - isomer) . reaction in vivo advantageously expands the scope of trans FIG . 40 illustrates the proposed synthesis of bicifadine , formations that are accessible to microbial organic synthesis DOV- 216 ,303 and derivatives using P450 - catalyzed cyclo and provides artificial metabolic pathways to complement propanation . 45 nature ' s existing strategies for making cyclopropanes . FIG . 41 illustrates that resting E . coli cells (425 uL ) were purged with argon , before adding glucose ( 50 uL ) , 2 , 5 II . Definitions dimethyl- 2 , 4 -hexadiene ( 12 . 5 uL ) , and EDA ( 12 . 5 uL ) . The reaction was carried out in nitrogen free M9 minimalmedia The following definitions and abbreviations are to be used with 5 % methanol cosolvent for 24 hours at 298 K . 50 for the interpretation of the invention . The term “ invention ” FIG . 42 illustrates a GC - FID chromatogram showing the or present invention ” as used herein is a non - limiting term two ethyl chrysanthemate diastereomers produced by BM3 - and is not intended to refer to any single embodiment but T268A - C400S . Oven temperature : 90° C . for 2 min , then 2º encompasses all possible embodiments . C ./ min to 110° C ., then 30° C . /min to 230° C . HP - 5 column As used herein , the terms " comprises, " " comprising , " ( Agilent ) 30 mx0 . 32 mmx0 . 25 um . 55 “ includes ,” “ including ," " has, " " having , " contains, " " con FIG . 43 illustrates a GC -MS ion chromatogram ( m = 123 ) taining , " or any other variation thereof, are intended to cover showing the two chrysanthemate diastereomers produced by a non -exclusive inclusion . A composition , mixture , process , BM3- T268A -C400S . Top insert shows the fragmentation method , article , or apparatus that comprises a list of ele pattern for ethyl chrysanthemate that gives rise to the ments is not necessarily limited to only those elements but molecular ion with m = 123 . Oven temperature : 90° C . for 2 60 may include other elements not expressly listed or inherent min , then 2° C ./ min to 110° C ., then 30° C ./ min to 230° C . to such composition , mixture , process, method , article, or HP - 5 column ( Agilent) 30 mx0 . 32 mmx0 .25 um . apparatus . Further , unless expressly stated to the contrary , FIG . 44 illustrates the chemical or enzymatic conversion " or” refers to an inclusive “ or ” and not to an exclusive " or ." of ethyl chrysanthemate to pyrethoid and pyrethrin insecti- The term " cyclopropanation ( enzyme) catalyst " or cides . (a ) Hydrolysis ; ( b ) activation to the acid chloride; ( c ) 65 " enzyme with cyclopropanation activity ” refers to any and coupling of the pyrethrolone alcohol (ROH ) to the acid all chemical processes catalyzed by enzymes , by which chloride ; ( d ) transesterification . substrates containing at least one carbon - carbon double US 10 , 208, 322 B2 21 bond can be converted into cyclopropane products by using than about 25 uM , preferably less than about 5 ?M , and even diazo reagents as carbene precursors . more preferably less than 1 uM . The term is also intended to The terms “ engineered heme enzyme” and “ heme enzyme include sealed chambers of liquid or solid medium main variant” include any heme- containing enzyme comprising at tained with an atmosphere of less than about 1 % oxygen . least one amino acid mutation with respect to wild - type and 5 Preferably , anaerobic conditions are achieved by sparging a also include any chimeric protein comprising recombined reaction mixture with an inert gas such as nitrogen or argon . sequences or blocks of amino acids from two , three , ormore As used herein , the term " exogenous ” is intended to mean different heme - containing enzymes . that the referenced molecule or the referenced activity is The terms " engineered cytochrome P450 ” and introduced into the host microbial organism . The term as it " cytochrome P450 variant” include any cytochrome P450 10 is used in reference to expression of an encoding nucleic enzyme comprising at least one amino acid mutation with acid refers to the introduction of the encoding nucleic acid respect to wild -type and also include any chimeric protein in an expressible form into the microbial organism . When comprising recombined sequences or blocks of amino acids used in reference to a biosynthetic activity , the term refers to from two , three, or more different cytochrome P450 an activity that is introduced into the host reference organ enzymes . 15 ism . The term “ whole cell catalyst ” includes microbial cells The term " heterologous” as used herein with reference to expressing heme -containing enzymes , wherein the whole molecules , and in particular enzymes and polynucleotides, cell catalyst displays cyclopropanation activity . indicates molecules that are expressed in an organism other As used herein , the terms “ porphyrin ” and “ metal- substi - than the organism from which they originated or are found tuted porphyrins ” include any porphyrin that can be bound 20 in nature, independently of the level of expression that can by a heme enzyme or variant thereof. In particular embodi- be lower , equal or higher than the level of expression of the ments , these porphyrins may contain metals including, but molecule in the native microorganism . not limited to , Fe, Mn, Co , Cu , Rh , and Ru . On the other hand , the term “ native ” or “ endogenous ” as The terms “ carbene equivalent” and “ carbene precursor” used herein with reference to molecules , and in particular include molecules that can be decomposed in the presence of 25 enzymes and polynucleotides , indicates molecules that are metal ( or enzyme) catalysts to structures that contain at least expressed in the organism in which they originated or are one divalent carbon with only 6 valence shell electrons and found in nature, independently of the level of expression that that can be transferred to C = C bonds to form cyclopropanes can be lower equal or higher than the level of expression of or to C - H or heteroatom - H bonds to form various carbon the molecule in the native microorganism . It is understood ligated products . 30 that expression of native enzymes or polynucleotides may be The terms " carbene transfer” and “ formal carbene trans - modified in recombinant microorganisms. fer " as used herein include any chemical transformation The term " homolog ," as used herein with respect to an where carbene equivalents are added to C = C bonds, car - original enzyme or gene of a first family or species, refers to bon -heteroatom double bonds or inserted into C / H or distinct enzymes or genes of a second family or species heteroatom - H substrates . 35 which are determined by functional, structural or genomic As used herein , the terms “ microbial, ” “ microbial organ analyses to be an enzyme or gene of the second family or ism ” and “ microorganism ” include any organism that exists species which corresponds to the original enzyme or gene of as a microscopic cell that is included within the domains of the first family or species . Homologs most often have archaea , bacteria or eukarya . Therefore, the term is intended functional , structural , or genomic similarities . Techniques to encompass prokaryotic or eukaryotic cells or organisms 40 are known by which homologs of an enzyme or gene can having a microscopic size and includes bacteria , archaea and readily be cloned using genetic probes and PCR . Identity of eubacteria of all species as well as eukaryotic microorgan cloned sequences as homolog can be confirmed using func isms such as yeast and fungi. Also included are cell cultures tional assays and / or by genomic mapping of the genes . of any species that can be cultured for the production of a protein has “ homology ” or is “ homologous” to a second chemical. 45 protein if the amino acid sequence encoded by a gene has a As used herein , the term " non - naturally occurring " , when similar amino acid sequence to that of the second gene . used in reference to a microbial organism or enzyme activity Alternatively , a protein has homology to a second protein if of the invention , is intended to mean that the microbial the two proteins have “ similar ” amino acid sequences . Thus , organism or enzyme has at least one genetic alteration not the term " homologous proteins " is intended to mean that the normally found in a naturally occurring strain of the refer - 50 two proteins have similar amino acid sequences . In particu enced species , including wild - type strains of the referenced lar embodiments , the homology between two proteins is species. Genetic alterations include , for example, modifica - indicative of its shared ancestry , related by evolution . tions introducing expressible nucleic acids encoding meta - The terms " analog ” and “ analogous ” include nucleic acid bolic polypeptides , other nucleic acid additions, nucleic acid or protein sequences or protein structures that are related to deletions and /or other functional disruption of the microbial 55 one another in function only and are not from common organism ' s genetic material. Such modifications include , for descent or do not share a common ancestral sequence . example , coding regions and functional fragments thereof, Analogs may differ in sequence but may share a similar for heterologous , homologous or both heterologous and structure , due to convergent evolution . For example , two homologous polypeptides for the referenced species. Addi- enzymes are analogs or analogous if the enzymes catalyze tionalmodifications include , for example , non - coding regu - 60 the same reaction of conversion of a substrate to a product, latory regions in which the modifications alter expression of are unrelated in sequence , and irrespective of whether the a gene or operon . Exemplary non - naturally occurring micro - two enzymes are related in structure . bial organism or enzyme activity includes the cyclopropa - As used herein , the term “ alkyl ” refers to a straight or nation activity described above . branched , saturated, aliphatic radical having the number of As used herein , the term “ anaerobic ” , when used in 65 carbon atoms indicated . Alkyl can include any number of reference to a reaction , culture or growth condition , is carbons , such as C1- 2 , C1- 3 , C1- 4 , C1- 5 , C1- 6 , C1- 7 , C1- 8 , C2- 3 , intended to mean that the concentration of oxygen is less C2- 4 , C2- 5 , C2- 6 , C3- 4 , C3- 5 , C3- 6 , C4- 5 , C4- 6 and C5- 6 . For US 10 ,208 , 322 B2 23 24 example , C1- 6 alkyl includes , but is not limited to , methyl, mantane. Cycloalkyl groups can also be partially unsatu ethyl, propyl, isopropyl, butyl, isobutyl, sec -butyl , tert -butyl , rated , having one or more double or triple bonds in the ring . pentyl, isopentyl, hexyl, etc . Alkyl can refer to alkyl groups Representative cycloalkyl groups that are partially unsatu having up to 20 carbons atoms, such as , but not limited to rated include, but are not limited to , cyclobutene , cyclopen heptyl , octyl, nonyl, decyl, etc . Alkyl groups can be option - 5 tene, cyclohexene, cyclohexadiene ( 1 , 3 - and 1 , 4 - isomers ) , ally substituted with one or more moieties selected from cycloheptene , cycloheptadiene , cyclooctene , cyclooctadiene halo , hydroxy, amino , alkylamino , alkoxy, haloalkyl, car- (1 ,3 -, 1 ,4 - and 1, 5 - isomers ), norbornene, and norbornadiene. boxy , amido , nitro , oxo , and cyano . Cycloalkyl groups can be optionally substituted with one or As used herein , the term “ alkenyl ” refers to a straight more moieties selected from halo , hydroxy, amino , alky chain or branched hydrocarbon having at least 2 carbon 10 lamino , alkoxy , haloalkyl, carboxy , amido , nitro , oxo , and atoms and at least one double bond . Alkenyl can include any cyano . number of carbons , such as C2, C2- 3 , C2- 4 , C2- 5 , C2- 6 , C2- 79 As used herein , the term " heterocyclyl ” refers to a satu C2- 8 , C2- 9 , C2- 10 , C3, C3- 4 , C3- 5 , C3- 6 , C4, C4- 5, C4- 6 , C5, rated ring system having from 3 to 12 ring members and C5 -6 , and Co . Alkenyl groups can have any suitable number from 1 to 4 heteroatoms selected from N , O and S . Addi of double bonds, including , but not limited to , 1 , 2 , 3 , 4 , 5 15 tional heteroatoms including , but not limited to , B , A1, Si and or more . Examples of alkenyl groups include , but are not P can also be present in a heterocycloalkyl group . The limited to , vinyl ( ethenyl) , propenyl, isopropenyl, 1 - butenyl, heteroatoms can be oxidized to form moieties such as, but 2 -butenyl , isobutenyl, butadienyl, 1 -pentenyl , 2 - pentenyl, not limited to , - S ( O ) - and - S ( O ) 2 — . Heterocyclyl isopentenyl , 1 , 3 - pentadienyl, 1 , 4 -pentadienyl , 1 -hexenyl , groups can include any number of ring atoms, such as, 3 to 2 - hexenyl, 3 -hexenyl , 1 , 3 -hexadienyl , 1 , 4 -hexadienyl , 1 , 5 - 20 6 , 4 to 6 , 5 to 6 , 4 to 6 , or 4 to 7 ring members. Any suitable hexadienyl, 2 , 4 -hexadienyl , or 1 , 3 , 5 - hexatrienyl. Alkenyl number of heteroatoms can be included in the heterocyclyl groups can be optionally substituted with one or more groups, such as 1 , 2 , 3 , or 4 , or 1 to 2 , 1 to 3 , 1 to 4 , 2 to 3 , moieties selected from halo , hydroxy , amino , alkylamino , 2 to 4 , or 3 to 4 . Examples of heterocyclyl groups include , alkoxy , haloalkyl, carboxy , amido , nitro , oxo , and cyano . but are not limited to , aziridine , azetidine, pyrrolidine , As used herein , the term “ alkynyl” refers to either a 25 piperidine , azepane , azocane , quinuclidine , pyrazolidine , straight chain or branched hydrocarbon having at least 2 imidazolidine, piperazine ( 1 , 2 - , 1 , 3 - and 1 , 4 - isomers ), oxi carbon atoms and at least one triple bond . Alkynyl can rane , oxetane , tetrahydrofuran , oxane (tetrahydropyran ) , include any number of carbons, such as C2, C2- 3, C2- 4, C2- 52 oxepane , thiirane , thietane, thiolane ( tetrahydrothiophene ), C2- 6 , C2- 7, C2 -3 , C2- 9, C2- 10 , C3, C3- 4 , C3- 5, C3- 6 , C4, C4- 59 thiane (tetrahydrothiopyran ), oxazolidine , isoxazolidine , thi C4- 6 , C5, C5- 6 , and Cg. Examples of alkynyl groups include, 30 azolidine, isothiazolidine , dioxolane , dithiolane, morpho but are not limited to , acetylenyl, propynyl , 1 -butynyl , line , thiomorpholine, dioxane , or dithiane . Heterocycly1 2 -butynyl , isobutynyl, sec- butynyl, butadiynyl , 1 - pentynyl, groups can be optionally substituted with one or more 2 - pentynyl, isopentynyl, 1 , 3 -pentadiynyl , 1 , 4 -pentadiynyl , moieties selected from halo , hydroxy, amino , alkylamino , 1 - hexynyl, 2 -hexynyl , 3 - hexynyl, 1 , 3 -hexadiynyl , 1 , 4 - alkoxy, haloalkyl , carboxy , amido , nitro , oxo , and cyano . hexadiynyl, 1 , 5 -hexadiynyl , 2 , 4 -hexadiynyl , or 1 , 3 , 5 - 35 As used herein , the term “ heteroaryl” refers to a mono hexatriynyl. Alkynyl groups can be optionally substituted cyclic or fused bicyclic or tricyclic aromatic ring assembly with one or more moieties selected from halo , hydroxy , containing 5 to 16 ring atoms, where from 1 to 5 of the ring amino , alkylamino , alkoxy, haloalkyl, carboxy, amido , nitro atoms are a heteroatom such as N , O or S . Additional oxo , and cyano . heteroatoms including , but not limited to , B , A1, Si and P can As used herein , the term “ aryl” refers to an aromatic 40 also be present in a heteroaryl group . The heteroatoms can carbon ring system having any suitable number of ring be oxidized to form moieties such as , but not limited to , atoms and any suitable number of rings . Aryl groups can — S ( O ) - and - S ( O ) , — Heteroaryl groups can include include any suitable number of carbon ring atoms, such as, any number of ring atoms, such as, 3 to 6 , 4 to 6 , 5 to 6 , 3 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or 16 ring atoms, as well as to 8 , 4 to 8 , 5 to 8 , 6 to 8 , 3 to 9 , 3 to 10 , 3 to 11 , or 3 to from 6 to 10 , 6 to 12 , or 6 to 14 ring members . Aryl groups 45 12 ring members . Any suitable number of heteroatoms can can be monocyclic , fused to form bicyclic or tricyclic be included in the heteroaryl groups , such as 1 , 2 , 3 , 4 , or 5 , groups, or linked by a bond to form a biaryl group . Repre or 1 to 2 , 1 to 3 , 1 to 4 , 1 to 5 , 2 to 3 , 2 to 4 , 2 to 5 , 3 to 4 , sentative aryl groups include phenyl, naphthyl and biphenyl. or 3 to 5 . Heteroaryl groups can have from 5 to 8 ring Other aryl groups include benzyl , having a methylene link - members and from 1 to 4 heteroatoms, or from 5 to 8 ring ing group . Some aryl groups have from 6 to 12 ring 50 members and from 1 to 3 heteroatoms, or from 5 to 6 ring members , such as phenyl, naphthyl or biphenyl . Other aryl members and from 1 to 4 heteroatoms, or from 5 to 6 ring groups have from 6 to 10 ring members , such as phenyl or members and from 1 to 3 heteroatoms. Examples of het naphthyl . Some other aryl groups have 6 ringmembers , such eroaryl groups include , but are not limited to , pyrrole , as phenyl . Aryl groups can be optionally substituted with pyridine , imidazole , pyrazole , triazole , tetrazole , pyrazine , one or more moieties selected from halo , hydroxy , amino , 55 pyrimidine, pyridazine , triazine ( 1 , 2 , 3 -, 1 , 2 , 4 - and 1 , 3 , 5 alkylamino , alkoxy , haloalkyl, carboxy, amido , nitro , oxo , isomers ) , thiophene , furan , thiazole , isothiazole , oxazole , and cyano . and isoxazole . Heteroaryl groups can be optionally substi As used herein , the term " cycloalkyl” refers to a saturated tuted with one ormore moieties selected from halo , hydroxy , or partially unsaturated , monocyclic , fused bicyclic or amino , alkylamino , alkoxy, haloalkyl, carboxy, amido, nitro , bridged polycyclic ring assembly containing from 3 to 12 60 Oxo , and cyano . ring atoms, or the number of atoms indicated . Cycloalkyl As used herein , the term “ alkoxy” refers to an alkyl group can include any number of carbons , such as C3- 6 , C4- 6 , C5- 6 , having an oxygen atom that connects the alkyl group to the C3- 8, C4- 8, C5 -8 , and C6- 8 . Saturated monocyclic cycloalkyl point of attachment: i. e . , alkyl- 0 — . As for alkyl group , rings include , for example , cyclopropyl, cyclobutyl, cyclo - alkoxy groups can have any suitable number of carbon pentyl , cyclohexyl, and cyclooctyl. Saturated bicyclic and 65 atoms, such as C1- 6 or C1- 4 . Alkoxy groups include, for polycyclic cycloalkyl rings include, for example , norbor example , methoxy , ethoxy, propoxy, iso - propoxy , butoxy, nane , [ 2 . 2 . 2 ]bicyclooctane , decahydronaphthalene and ada - 2 -butoxy , iso -butoxy , sec - butoxy, tert - butoxy, pentoxy , US 10 , 208 , 322 B2 25 26 hexoxy , etc . Alkoxy groups can be optionally substituted compounds belonging to these and other classes ( e . g ., chry with one or more moieties selected from halo , hydroxy , santhemate esters for the synthesis of pyrethroid com amino , alkylamino , alkoxy, haloalkyl, carboxy, amido , nitro , pounds ) . oxo , and cyano. In other aspects , the present invention provides heme As used herein , the term “ alkylthio ” refers to an alkyl 5 enzymes including variants and fragments thereof (e . g. , group having a sulfur atom that connects the alkyl group to truncated forms ) as well as chimeric heme enzymes that are the point of attachment: i. e ., alkyl- S — . As for alkyl groups , capable of carrying out the cyclopropanation reactions alkylthio groups can have any suitable number of carbon described herein . Expression vectors and host cells express exampleatoms, such , methoxy as C1- , 6 ethoxyor C1- ,4 .propoxy Alkylthio , iso groups - propoxy include . butoxv , for . 10 ing the heme enzymes are also provided by the present 2 -butoxy , iso -butoxy , sec -butoxy , tert- butoxy, pentoxy, invention . hexoxy, etc . Alkylthio groups can be optionally substituted The following sections provide a description of exem with one or more moieties selected from halo , hydroxy , plary and preferred embodiments including heme enzymes , amino , alkylamino, alkoxy, haloalkyl, carboxy , amido , nitro , expression vectors , host cells , cyclopropanation products oxo , and cyano . 15 such as, e . g ., compounds comprising one or more cyclopro As used herein , the terms “ halo ” and “ halogen ” refer to pane functional groups , starting materials such as , e . g. , fluorine, chlorine, bromine and iodine. olefinic substrates and diazo reagents , and characteristics As used herein , the term “ haloalkyl” refers to an alkyl and reaction conditions for the in vitro and in vivo cyclo moiety as defined above substituted with at least one halo gen atom . 20 propanation reactions described herein . As used herein , the term “ alkylsilyl” refers to a moietybiety A . Heme Enzymes — SiRz, wherein at least one R group is alkyl and the other In certain aspects , the present invention provides compo R groups are H or alkyl . The alkyl groups can be substituted sitions comprising one or more heme enzymes that catalyze with one more halogen atoms. the conversion of an olefinic substrate to products containing As used herein , the term “ acy?” refers to a moiety - C ( O ) 25 one or more cyclopropane functional groups. In particular R , wherein R is an alkyl group . embodiments , the present invention provides heme enzyme As used herein , the term " oxo ” refers to an oxygen atom that is double - bonded to a compound ( i . e . , OS ) . variants comprising at least one or more amino acid muta As used herein , the term “ carboxy ” refers to a moiety tions therein that catalyze the formal transfer of carbene - C (O )OH . The carboxy moiety can be ionized to form the 30 equivalents from a diazo reagent ( e . g . , a diazo ester ) to an carboxylate anion . olefinic substrate , making cyclopropane products with high As used herein , the term “ amino” refers to a moiety stereoselectivity . In preferred embodiments , the heme - NR2, wherein each R group is H or alkyl. enzyme variants of the present invention have the ability to As used herein , the term “ amido ” refers to a moiety catalyze cyclopropanation reactions efficiently , display - NRC ( O )R or - C (O )NR2 , wherein each R group isSH H or 3535 metincreased total turnover numbers , and /or demonstrate highly alkyl. regio - and / or enantioselective product formation compared to the corresponding wild - type enzymes . III . Description of the Embodiments The terms “ heme enzyme” and “ heme protein ” are used herein to include any member of a group of proteins con In some aspects, the present invention provides methods 40 taining heme as a prosthetic group . Non - limiting examp for catalyzing the conversion of an olefin to any compound ( e . g . , any intermediate or final compound ) containing one or of heme enzymes include globins, cytochromes , oxi more cyclopropane functional groups using heme enzymes. doreductases, any other protein containing a heme as a In certain aspects , the present invention provides a method prosthetic group , and combinations thereof. Heme- contain for producing a cyclopropanation product, the method com - 45 ing globins include , but are not limited to , hemoglobin , prising : myoglobin , and combinations thereof. Heme - containing ( a ) providing an olefinic substrate , a diazo reagent, and a cytochromes include , but are not limited to , cytochrome heme enzyme; and P450 , cytochrome b , cytochrome cl , cytochrome c , and ( b ) admixing the components of step ( a ) in a reaction for combinations thereof. Heme - containing oxidoreductases a time sufficient to produce a cyclopropanation product. 50 include , but are not limited to , a catalase , an oxidase , an In certain instances , the cyclopropanation product is pro oxygenase , a haloperoxidase , a peroxidase , and combina duced via an intermolecular cyclopropanation reaction tions thereof. between the olefinic substrate and diazo reagent as separate In certain instances, the heme enzymes are metal- substi distinct substrates . In other instances , the cyclopropanation tuted heme enzymes containing protoporphyrin IX or other product is produced via an intramolecular cyclopropanation 55 porphyrin molecules containing metals other than iron , reaction , e . g . , wherein the olefinic substrate and diazo including , but not limited to , cobalt , rhodium , copper , ruthe reagent are part of the same substrate . nium , and manganese , which are active cyclopropanation The methods of the invention can be used to provide a catalysts . vast number of cyclopropanation products. The cyclopropa - In some embodiments , the heme enzyme is a member of nation products include classes of compounds such as , but 60 one of the enzyme classes set forth in Table 1 . In other not limited to , insecticides ( e . g . , pyrethroid compounds ) , embodiments , the heme enzyme is a variant or homolog of active pharmaceutical agents having chiral and /or achiral a member of one of the enzyme classes set forth in Table 1. cyclopropane moieties ( e . g . , milnacipran , levomilnacipran , In yet other embodiments , the heme enzyme comprises or and other active ingredients such as antibiotics , antivirals , consists of the heme domain of a member of one of the etc . ), commodity and fine chemicals , plant hormones, fla - 65 enzyme classes set forth in Table 1 or a fragment thereof vors and scents , and fatty acids. The cyclopropanation ( e . g ., a truncated heme domain ) that is capable of carrying products can also serve as intermediates for the synthesis of out the cyclopropanation reactions described herein . US 10 ,208 , 322 B2 27 28 TABLE 1 TABLE 1 -continued Heme enzymes identified by their enzyme classification Heme enzymes identified by their enzyme classification number (EC number) and classification name. number (EC number) and classification name. EC Number Name 5 EC Number Name 1 . 1. 2 . 3 L -lactate dehydrogenase 1 . 11 . 2 . 1 unspecific peroxygenase 1 . 1 . 2 . 6 polyvinyl alcohol dehydrogenase ( cytochrome ) 1 . 11. 2 . 2 myeloperoxidase 1 . 1 . 2 . 7 methanol dehydrogenase ( cytochrome c ) 1. 11 . 2 . 3 plant seed peroxygenase 1 . 1 . 5 . 5 alcohol dehydrogenase ( quinone ) 1 . 11 . 2 . 4 fatty - acid peroxygenase 1 . 1 . 5 . 6 formate dehydrogenase - N : 10 1 . 12 . 2 . 1 cytochrome- c3 hydrogenase 1 . 1 . 9 . 1 alcohol dehydrogenase (azurin ) : 1 . 12 . 5 . 1 hydrogen : quinone oxidoreductase 1 . 1 . 99 . 3 gluconate 2 - dehydrogenase (acceptor ) 1 . 12. 99 . 6 hydrogenase (acceptor ) 1 . 1 . 99 . 11 fructose 5 - dehydrogenase 1 . 13 . 11 . 9 2 ,5 - dihydroxypyridine 5 , 6 -dioxygenase 1 . 1 . 99 . 18 cellobiose dehydrogenase ( acceptor ) 1 . 13 . 11. 11 tryptophan 2 , 3 - dioxygenase 1 . 1 . 99 . 20 alkan - 1 -ol dehydrogenase ( acceptor ) 1 . 13 . 11 . 49 chlorite O2 - lyase 1 . 2 . 1 . 70 glutamyl -tRNA reductase 15 1 .13 . 11 . 50 acetylacetone -cleaving enzyme 1 . 2 . 3 . 7 indole - 3 -acetaldehyde oxidase 1 . 13 . 11 . 52 indoleamine 2 , 3 -dioxygenase 1 . 2 . 99 . 3 aldehyde dehydrogenase (pyrroloquinoline -quinone ) 1 . 13. 11. 60 linoleate 8R - lipoxygenase 1 . 3 . 1 . 6 fumarate reductase (NADH ) : 1 . 13. 99 . 3 tryptophan 2 '- dioxygenase 1 . 3 . 5 . 1 succinate dehydrogenase (ubiquinone ) 1 . 14 . 11 . 9 flavanone 3 -dioxygenase 1 . 3 . 5 . 4 fumarate reductase (menaquinone ) 1 . 14 . 12 . 17 nitric oxide dioxygenase 1 . 3 . 99 . 1 succinate dehydrogenase 1 . 14 . 13 . 39 nitric -oxide synthase (NADPH dependent ) 1 . 4 . 9 . 1 methylamine dehydrogenase (amicyanin ) 20 1 . 14 . 13 . 17 cholesterol Talpha -monooxygenase 1 . 4 . 9 . 2 . aralkylamine dehydrogenase (azurin ) 1 . 14 . 13 .41 tyrosine N -monooxygenase 1 . 5 . 1 . 20 methylenetetrahydrofolate reductase [NAD ( P ) H ] 1. 14 . 13 . 70 sterol 14alpha - demethylase 1 . 5 . 99 . 6 spermidine dehydrogenase 1 . 14 . 13 . 71 N -methylcoclaurine 3 '- monooxygenase 1 . 6 . 3 . 1 NAD ( P ) H oxidase 1 . 14 . 13. 81 magnesium -protoporphyrin IX monomethyl ester 1 . 7 . 1 . 1 nitrate reductase (NADH ) ( oxidative ) cyclase 1 . 7 . 1 . 2 Nitrate reductase [NAD ( P ) H ] 25 1 . 14 .13 . 86 2 -hydroxyisoflavanone synthase 1 . 7 . 1 . 3 nitrate reductase (NADPH ) 1 . 14 . 13 . 98 cholesterol 24 -hydroxylase 1 . 7 . 1 . 4 nitrite reductase [NAD ( P ) H ] 1 . 14 . 13. 119 5 -epiaristolochene 1 ,3 -dihydroxylase 1 . 7 . 1 . 14 nitric oxide reductase [NAD ( P ) , nitrous oxide- forming ] 1 . 14 . 13 . 126 vitamin D3 24 -hydroxylase 1 . 7 . 2 . 1 nitrite reductase (NO - forming ) 1 . 14 . 13 . 129 beta -carotene 3 -hydroxylase 1 . 7 . 2 . 2 nitrite reductase ( cytochrome; ammonia - forming) 1 . 14 . 13 . 141 cholest- 4 - en - 3 -one 26 -monooxygenase 1 . 7 . 2 . 3 trimethylamine - N -oxide reductase (cytochrome c ) 30 1 . 14 .13 . 142 3 - ketosteroid Salpha -monooxygenase 1 . 7 . 2 . 5 nitric oxide reductase (cytochrome c ) 1 . 14 . 13 . 151 linalool 8 -monooxygenase 1 . 7 . 2 . 6 hydroxylamine dehydrogenase 1 . 14 . 13. 156 1, 8 - cineole 2 - endo -monooxygenase 1 . 7 . 3 . 6 hydroxylamine oxidase ( cytochrome ) 1 . 14 . 13 . 159 vitamin D 25 - hydroxylase 1 . 7 . 5 . 1 nitrate reductase ( quinone ) 1 . 14 . 14 . 1 unspecific monooxygenase 1 . 7 . 5 . 2 nitric oxide reductase (menaquinol ) 1 . 14 . 15 . 1 camphor 5 -monooxygenase 1 . 7 . 6 . 1 nitrite dismutase 35 1. 14. 15. 6 cholesterol monooxygenase (side - chain -cleaving ) 1 . 7 . 7 . 1 ferredoxin -nitrite reductase 1 . 14 . 15 . 8 steroid 15beta -monooxygenase 1 . 7 . 7 . 2 fenedoxin - nitrate reductase 1 . 14 . 15 . 9 spheroidene monooxygenase 1 . 7 . 99 . 4 nitrate reductase 1 . 14 . 18 . 1 tyrosinase 1 . 7 . 99 . 8 hydrazine oxidoreductase 1 . 14 . 19 . 1 stearoyl - CoA 9 - desaturase 1 . 8 . 1 . 2 sulfite reductase (NADPH ) 1 . 14 . 19 . 3 linoleoyl - CoA desaturase 1 . 8 . 2 . 1 sulfite dehydrogenase 1 . 14 . 21 . 7 biflaviolin synthase 1 . 8 . 2 . 2 thiosulfate dehydrogenase 40 1 . 14 . 99 . 1 prostaglandin - endoperoxide synthase 1 . 8 . 2 . 3 sulfide - cytochrome- c reductase ( flavocytochrome c ) 1 . 14 . 99 . 3 heme oxygenase 1 . 8 . 2 . 4 dimethyl sulfide :cytochrome c2 reductase 1 . 14 .99 . 9 steroid 17alpha- monooxygenase 1 . 8 . 3 . 1 sulfite oxidase 1 . 14 . 99 . 10 steroid 21 -monooxygenase 1 . 8 . 7 . 1 sulfite reductase ( ferredoxin ) 1. 14 .99 . 15 4 -methoxybenzoate monooxygenase ( O - demethylating ) 1 . 8 . 98 . 1 CoB - COM heterodisulfide reductase 1 . 14 . 99 . 45 carotene epsilon -monooxygenase 1 . 8 . 99 . 1 sulfite reductase 45 1 . 16 . 5 . 1 ascorbate ferrireductase ( transmembrane ) 1 . 8 .99 . 2 adenylyl -sulfate reductase 1 . 16 . 9 . 1 iron : rusticyanin reductase 1 . 8 . 99. 3 hydrogensulfite reductase 1 . 17 . 1 . 4 xanthine dehydrogenase 1 . 9 . 3 . 1 cytochrome - c oxidase 1 . 17 . 2 . 2 lupanine 17 -hydroxylase (cytochrome c) 1 . 9 . 6 . 1 nitrate reductase ( cytochrome) 1 . 17 . 99 . 1 4 -methylphenol dehydrogenase (hydroxylating ) 1 . 10 . 2 . 2 ubiquinol- cytochrome- c reductase 1 . 17 . 99 . 2 ethylbenzene hydroxylase 1 . 10 . 3 . 1 catechol oxidase 50 1. 97 . 1 . 1 chlorate reductase 1 . 10 . 3 . B1 caldariellaquinol oxidase ( H + - transporting ) 1 . 97 . 1 . 9 selenate reductase 1 . 10 . 3 . 3 L - ascorbate oxidase 2 . 7 . 7 .65 diguanylate cyclase 1 . 10 . 3 . 9 photosystem II 2 . 7 . 13. 3 histidine kinase 1 . 10 . 3 . 10 ubiquinol oxidase ( H + - transporting ) 3 . 1 . 4 . 52 cyclic - guanylate - specific phosphodiesterase 1 . 10 . 3 . 11 ubiquinol oxidase 4 . 2 . 1 . B9 colneleic acid / etheroleic acid synthase 1 . 10 . 3 . 12 menaquinol oxidase ( H + -transporting ) 4 . 2 . 1 . 22 Cystathionine beta -synthase 1 . 10 . 9 . 1 plastoquinol- plastocyanin reductase 55 4 . 2 . 1. 92 hydroperoxide dehydratase 1 . 11 . 1 . 5 cytochrome- c peroxidase 4 . 2 . 1 . 212 colneleate synthase 1 . 11 . 1 . 6 catalase 4 . 3 . 1 . 26 chromopyrrolate synthase 1 . 11 . 1 . 7 peroxidase 4 . 6 . 1 . 2 guanylate cyclase 1 . 11 . 1 . B2 chloride peroxidase ( vanadium - containing ) 4 . 99 . 1 . 3 sirohydrochlorin cobaltochelatase 1 . 11 . 1 . B7 bromide peroxidase ( heme- containing ) 4 . 99 . 1 . 5 aliphatic aldoxime dehydratase 1 . 11 . 1 . 8 iodide peroxidase 60 4 . 99 . 1 . 7 phenylacetaldoxime dehydratase 1 . 11 . 1 . 10 chloride peroxidase 5 . 3 . 99 . 3 prostaglandin - E synthase 1 . 11 . 1 . 11 L -ascorbate peroxidase 5 . 3 .99 . 4 prostaglandin - I synthase 1 . 11 . 1 . 13 manganese peroxidase 5 . 3 . 99 .5 Thromboxane - A synthase 1 . 11 . 1 . 14 lignin peroxidase 5 . 4 . 4 . 5 9, 12 -octadecadienoate 8 - hydroperoxide 8R - isomerase 1 . 11 . 1 . 16 versatile peroxidase 5 . 4 . 4 . 6 9 ,12 -octadecadienoate 8 -hydroperoxide 8S - isomerase 1 . 11 . 1 . 19 dye decolorizing peroxidase 65 6 . 6 . 1 . 2 cobaltochelatase 1 . 11 . 1 . 21 catalase -peroxidase US 10 , 208 , 322 B2 29 30 In particular embodiments , the heme enzyme is a variant reductase domain or an accessory cytochrome P450 reduc or a fragment thereof ( e . g . , a truncated variant containing the tase enzyme, the heme center of cytochrome P450 activates heme domain ) comprising at least one mutation such as, e . g ., molecular oxygen , generating a high valent iron ( IV ) -oxo a mutation at the axial position of the heme coordination porphyrin cation radical species intermediate (Compound I , site . In some instances, the mutation is a substitution of the 5 FIG . 1 ) and a molecule of water . native residue with Ala , Asp , Arg , Asn , Cys, Glu , Gin , Gly , One skilled in the art will appreciate that the cytochrome His , Ile , Lys , Leu , Met , Phe , Pro , Ser, Thr, Trp , Tyr, or Val P450 enzyme superfamily has been compiled in various at the axial position . In certain instances , the mutation is a databases , including , but not limited to , the P450 homepage substitution of Cys with any other amino acid such as Ser at ( available at http : / /drnelson .uthsc .edu / the axial position . 1010 CytochromeP450C . html; see also , D . R . Nelson , Hum . In certain embodiments, the in vitro methods for produc Genomics 4 , 59 ( 2009 ) ), the cytochrome P450 enzyme ing a cyclopropanation product comprise providing a heme engineering database ( available at http :/ /www .cyped . uni enzyme, variant, or homolog thereof with a reducing agent stuttgart. de / cgi- bin / CYPED5/ index . pl; see also , D . Sirim et such as NADPH or a dithionite salt ( e . g ., Na2S204) . In 21. BMC Biochem 10 27 09009 )) and the Superrun data certain other embodiments , the in vivo methods for produc- 15 base ( available at http : / /bioinformatics . charite .de / supercyp / ; ing a cyclopropanation product comprise providing whole see also , S . Preissner et al. , Nucleic Acids Res. 38 , D237 cells such as E . coli cells expressing a heme enzyme , variant, (2010 ) ) , the disclosures of which are incorporated herein by or homolog thereof. reference in their entirety for all purposes . In some embodiments , the heme enzyme , variant, or In certain embodiments , the cytochrome P450 enzymes of homolog thereof is recombinantly expressed and optionally 20 the invention are members of one of the classes shown in isolated and / or purified for carrying out the in vitro cyclo Table 2 (see , http : / /www .icgeb .org / ~ p450srv / propanation reactions of the present invention . In other P450enzymes .html , the disclosure ofwhich is incorporated embodiments , the heme enzyme, variant, or homolog thereof is expressed in whole cells such as E . coli cells , and herein by reference in its entirety for all purposes) . these cells are used for carrying out the in vivo cyclopro - 25 panation reactions of the present invention . TABLE 2 In certain embodiments , the heme enzyme, variant , or Cytochrome P450 enzymes classified by their EC number , homolog thereof comprises or consists of the same number recommended name, and family / gene name. of amino acid residues as the wild - type enzyme ( e . g . , a Family / full -length polypeptide) . In some instances , the heme 30 EC Recommended name gene enzyme, variant, or homolog thereof comprises or consists 1 . 3 . 3 . 9 secologanin synthase CYP72A1 of an amino acid sequence without the start methionine ( e . g ., 1 . 14 . 13 . 11 trans- cinnamate 4 -monooxygenase CYP73 P450 BM3 amino acid sequence set forth in SEQ ID NO : 1 ) . 1 . 14 . 13 . 12 benzoate 4 -monooxygenase CYP53 In other embodiments , the heme enzyme comprises or 1. 14 . 13 . 13 calcidiol 1 -monooxygenase CYP27 consists of a heme domain fused to a reductase domain . In 35 1. 14 .13 .15 cholestanetriol 26 -monooxygenase CYP27 1 . 14 . 13 . 17 cholesterol 7a -monooxygenase CYP7 yet other embodiments , the heme enzyme does not contain 1 . 14 . 13 . 21 flavonoid 3 ' -monooxygenase CYP75 a reductase domain , e . g . , the heme enzyme contains a heme 1 . 14 . 13 .28 3 , 9 - dihydroxypterocarpan 6a -monooxygenase CYP93A1 domain only or a fragment thereof such as a truncated heme 1 . 14 .13 . 30 leukotriene- B4 20 -monooxygenase CYP4F domain . 1 . 14 .13 . 37 methyltetrahydroprotoberberine 14 CYP93A1 monooxygenase In some embodiments , the heme enzyme, variant, or 40 1 . 14 . 13. 41 tyrosine N -monooxygenase CYP79 homolog thereof has an enhanced cyclopropanation activity 1 . 14 . 13 .42 hydroxyphenylacetonitrile 2 -monooxygenase of at least about 1 . 5 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , 1 . 14 .13 . 47 ) - limonene 3 -monooxygenase 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , or 100 fold 1 . 14 . 13 . 48 (- ) - limonene 6 -monooxygenase 1. 14 . 13. 49 ( - ) - limonene 7 -monooxygenase compared to the corresponding wild - type heme enzyme. 1 .14 . 13 . 52 isoflavone 3 ' -hydroxylase | In some embodiments , the heme enzyme, variant, or 45 1. 14 .13 .53 isoflavone 2' -hydroxylase homolog thereof has a resting state reduction potential 1. 14 . 13. 55 protopine 6 -monooxygenase higher than that of NADH or NADPH . 1 . 14 . 13. 56 dihydrosanguinarine 10 -monooxygenase In particular embodiments , the heme enzyme comprises a 1 . 14 . 13. 57 dihydrochelirubine 12 -monooxygenase 1 . 14 . 13 .60 27 -hydroxycholesterol 7a -monooxygenase cyctochrome P450 enzyme. Cytochrome P450 enzymes 1. 14 . 13. 70 sterol 14 - demethylase CYP51 constitute a large superfamily of heme- thiolate proteins 50 1. 14 . 13. 71 N -methylcoclaurine 3 '- monooxygenase CYP80B1 involved in the metabolism of a wide variety of both 1 . 14 .13 . 73 tabersonine 16 - hydroxylase CYP71D12 exogenous and endogenous compounds . Usually , they act as 1 . 14 . 13 .74 7 -deoxyloganin 7 -hydroxylase 1 . 14 . 13. 75 vinorine hydroxylase the terminal oxidase in multicomponent electron transfer 1 . 14 . 13. 76 taxane 10B -hydroxylase CYP725A1 chains, such as P450 -containing monooxygenase systems. 1 . 14 . 13. 77 taxane 13a -hydroxylase CYP725A2 Members of the cytochrome P450 enzyme family catalyze 55 1 . 14 . 13. 78 ent- kaurene oxidase CYP701 1 . 14 . 13. 79 ent- kaurenoic acid oxidase CYP88A myriad oxidative transformations, including , e . g ., hydroxy 1 . 14 . 14 . 1 unspecific monooxygenase multiple lation , epoxidation , oxidative ring coupling , heteratom 1 . 14 . 15 . 1 camphor 5 -monooxygenase CYP101 release , and heteroatom oxygenation ( E . M . Isin et al. , 1 . 14 . 15 . 3 alkane 1 -monooxygenase CYP4A Biochim . Biophys. Acta 1770 , 314 (2007 )) . The active site of 1. 14 . 15 .4 steroid 11B -monooxygenase CYP11B actor co 1 . 14 . 15 . 5 corticosterone 18 -monooxygenase CYP11B these enzymes contains an Feth -protoporphyrin IX cofactor 60 1 . 14 . 15 . 6 cholesterol monooxygenase (side - chain -cleav - CYP11A (heme ) ligated proximally by a conserved cysteine thiolate ing ) ( M . T. Green , Current Opinion in Chemical Biology 13, 84 1 . 14 .21 . 1 ( S )- stylopine synthase ( 2009 )) . The remaining axial iron coordination site is occu 1 . 14 . 21. 2 ( S ) - cheilanthifoline synthase pied by a water molecule in the resting enzyme, but during 1 . 14 . 21 . 3 berbamunine synthase CYP80 native catalysis , this site is capable of binding molecular 65 1 . 14 . 21 . 54 salutaridine(S ) -canadine synthase synthase oxygen . In the presence of an electron source , typically 1 . 14 . 99. 9 steroid 17a -monooxygenase CYP17 provided by NADH or NADPH from an adjacent fused US 10 , 208 , 322 B2 31 32 TABLE 2 -continued 087381; WO 2003/ 078577 ; US 20030170627 ; US 20030166176 ; US 20030150025 ; WO 2003 /057830 ; WO Cytochrome P450 enzymes classified by their EC number , 2003/ 052050 ; CN 1358756 ; US 20030092658 ; US recommended name, and family / gene name. 20030078404 ; US 20030066103 ; WO 2003 /014341 ; US Family / 20030022334 ; WO 2003 / 008563 ; EP 1270722 ; US EC Recommended name gene 20020187538 ; WO 2002 /092801 ; WO 2002 /088341 ; US 1 . 14 .99 . 10 steroid 21 -monooxygenase CYP21 20020160950 ; WO 2002 /083868 ; US 20020142379 ; WO 1 . 14 . 99 .22 ecdysone 20 -monooxygenase 2002 /072758 ; WO 2002 /064765 ; US 20020076777 ; US 1 . 14 . 99 .28 linalool 8 -monooxygenase CYP111 20020076774 ; US 20020076774 ; WO 2002 /046386 ; WO 4 . 2 . 1 .92 hydroperoxide dehydratase CYP74 10 5 . 3. 99 .4 prostaglandin - I synthase CYP8 2002 /044213 ; US 20020061566 ; CN 1315335 ; WO 2002/ 5 . 3 . 99 . 5 thromboxane - A synthase CYP5 034922 ; WO 2002/ 033057 ; WO 2002/ 029018 ; WO 2002/ 018558 ; JP 2002058490 ; US 20020022254 ; WO 2002 / 008269; WO 2001/ 098461 ; WO 2001/ 081585 ; WO 2001/ Table 3 below lists additional cyctochromeP450 enzymes 051622 ; WO 2001/ 034780 ; CN 1271005 ; WO 2001/ that are suitable for use in the cyclopropanation reactions of 15 011071 ; WO 2001 /007630 ; WO 2001/ 007574 ; WO 2000 / the present invention . The accession numbers in Table 3 are 078973 ; U .S . Pat. No. 6 , 130 ,077 ; JP 2000152788 ; WO incorporated herein by reference in their entirety for all 2000 /031273 ; WO 2000 /020566 ; WO 2000 /000585 ; DE purposes . The cytochrome P450 gene and / or protein 19826821; JP 11235174 ; U . S . Pat. No. 5 , 939, 318 ; WO sequences disclosed in the following patent documents are 99 / 19493 ; WO 99 / 18224 ; U . S . Pat. No . 5 ,886 , 157 ; WO hereby incorporated by reference in their entirety for all 20 99 /08812 : US Pat No . 5 . 869. 283 : JP 10262665 : WO purposes: WO 2013 /076258 ; CN 103160521 ; CN 98 / 40470 ; EP 776974 ; DE 19507546 ; GB 2294692 ; U . S . 103223219 ; KR 2013081394 ; JP 5222410 ; WO 2013 / Pat . No . 5 ,516 ,674 ; JP 07147975 ; WO 94 /29434 ; JP 073775 ; WO 2013 /054890 ; WO 2013 /048898 ; WO 2013 / 06205685 ; JP 05292959 ; JP 04144680 ; DD 298820 ; EP 031975 ; WO 2013 / 064411 ; U .S . Pat . No . 8, 361, 769 ; WO 477961 ; SU 1693043 ; JP 01047375 ; EP 281245 ; JP 2012 / 150326 , CN 102747053 ; CN 102747052 ; JP 25 62104583 : IP 63044888 : IP 62236485 . IP 62104582 . and IP 2012170409 ; WO 2013 / 115484 ; CN 103223219 ; KR 2013081394 ; CN 103194461 ; JP 5222410 ; WO 2013 / 62019084 . 086499 ; WO 2013 /076258 ; WO 2013 /073775 ; WO 2013 / 064411 ; WO 2013 /054890 ; WO 2013 /031975 ; U . S . Pat. No . TABLE 3 8 . 361. 769: WO 2012 / 156976 ; WO 2012 / 150326 ; CN 30 Additional cytochrome P450 enzymes of the present invention . 102747053 ; CN 102747052 ; US 20120258938 ; JP 2012170409; CN 102399796 ; JP 2012055274 ; WO 2012/ Species Cyp No . Accession No . SEQ ID NO 029914 ; WO 2012 /028709 ; WO 2011/ 154523 ; JP Bacillus megaterium 102A1 AAA87602 ? 102A1 ADA57069 2011234631; WO 2011 / 121456 ; EP 2366782 ; WO 2011 / Bacillus megaterium ? 105241; CN 102154234 ; WO 2011 / 093185 ; WO 2011 / 35 Bacillus megaterium 102A1 ADA57068 Bacillus megaterium 102A1 ADA57062 ? 093187 ; WO 2011 /093186 ; DE 102010000168 ; CN Bacillus megaterium 102A1 ADA57061 102115757 ; CN 102093984 ; CN 102080069 ; JP Bacillus megaterium 102A1 ADA57059 ? 2011103864 ; WO 2011 /042143 ; WO 2011 /038313 ; JP Bacillus megaterium 102A1 ADA57058 2011055721; WO 2011 /025203 ; JP 2011024534 ; WO 2011 / Bacillus megaterium 102A1 ADA57055 ? Bacillus megaterium 102A1 ACZ37122 ? 008231; WO 2011/ 008232 ; WO 2011/ 005786 ; IN 40 BacillusB megaterium 102A1 ADA57057 ? 2009DE01216 ; DE 102009025996 ; WO 2010 / 134096 ; JP Bacillus megaterium 102A1 ADA57056 ? 2010233523 ; JP 2010220609 ; WO 2010 /095721 ; WO 2010 / Mycobacterium sp . HXN -1500 153A6 CAH04396 064764 ; US 20100136595 ; JP 2010051174 ; WO 2010 / Tetrahymena thermophile 5013C2 ABY59989 ????. Nonomuraea dietziae AGE14547 . 1 14 024437 ; WO 2010 /011882 ; WO 2009/ 108388 ; US Homo sapiens 2R1 NP 078790 20090209010 ; US 20090124515 ; WO 2009 /041470 ; KR 45 Macca mulatta 2R1 NP 001180887 . 1 16 2009028942 ; WO 2009/ 039487 ; WO 2009/ 020231 ; JP Canis familiaris 2R1 XP 854533 2009005687 ; CN 101333520 ; CN 101333521 ; US Mus musculus 2R1 AAIO8963 Bacillus halodurans C - 125 152A6 NP _ 242623 20080248545 ; JP 2008237110 ; CN 101275141 ; WO 2008 / Streptomyces parvus aryc AFM80022 118545 ; WO 2008 / 115844 ; CN 101255408 ; CN 101250506 ; Pseudomonas putida 101A1 P00183 CN 101250505 ; WO 2008 /098198 ; WO 2008/ 096695 ; WO 50 Homo sapiens 2D7 AAO49806 2008 /071673 ; WO 2008 / 073498 ; WO 2008 /065370 ; WO Rattus norvegicus C27 AABO2287 2008 /067070 ; JP 2008127301; JP 2008054644 ; KR 794395 ; Oryctolagus cuniculus 2B4 AAA65840 AWNO Bacillus subtilis 102A2 008394 EP 1881066 ; WO 2007 / 147827 ; CN 101078014 ; JP Bacillus subtilis 102A3 008336 2007300852 ; WO 2007 /048235 ; WO 2007 / 044688 ; WO B . megaterium DSM 32 102A1 P14779 2007 /032540 ; CN 1900286 ; CN 1900285 ; JP 2006340611 : 55 B . cereus ATCC14579 102A5 AP10153 B . licheniformis ATTC1458 102A7 YP 079990 WO 2006 / 126723 ; KR 2006029792 ; KR 2006029795 ; WO B . thuringiensis serovar YP 037304 30 2006 / 105082 ; WO 2006 / 076094 ; US 2006 /0156430 ; WO konkukian 2006 /065126 ; JP 2006129836 ; CN 1746293 ; WO 2006 / str . 97- 27 029398 ; JP 2006034215 ; JP 2006034214 ; WO 2006 / R . metallidurans CH34 102E1 YP 585608 A . fumigatus Af293 505X EAL92660 009334 ; WO 2005 / 111216 ; WO 2005 /080572 ; US 2005/ 60 A . nidulans FGSC A4 505A8 EAA58234 0150002 ; WO 2005 / 061699 ; WO 2005 /052152 ; WO 2005 / A . oryzae ATCC42149 505A3 Q2U4F1 038033 ; WO 2005 /038018 ; WO 2005 /030944 ; JP A . oryzae ATCC42149 X Q2UNA2 ?????? 2005065618 ; WO 2005 /017106 ; WO 2005 /017105 ; US F. oxysporum 505A1 Q9Y8G7 G . moniliformis X AAG27132 20050037411; WO 2005 /010166 ; JP 2005021106 ; JP G . zeae PH1 505A7 EAA67736 2005021104 ; JP 2005021105 ; WO 2004 / 113527 ; CN 65 G . zeae PH1 505C2 EAA77183 1472323 ; JP 2004261121; WO 2004/ 013339 ; WO 2004 M . grisea 70 - 15 syn 505A5 XP 365223 40Antona 011648 ; DE 10234126 ; WO 2004 / 003190 ; WO 2003 / US 10 , 208 , 322 B2 33 34 TABLE 3 -continued alignment algorithms may show a phylogenetic similarity between a P450 enzyme for which crystallographic or Additional cytochrome P450 enzymes of the present invention . mutagenesis data exist and a new P450 enzyme for which Species Cyp No. Accession No . SEQ ID NO such data do not exist. Thus, the polypeptide sequences of the present invention for which the heme axial ligand is N . crassa OR74 A 505A2 XP 961848 41 Oryza sativa * 97A known can be used as a “ query sequence” to perform a Oryza sativa * 97B search against a specific new cytochrome P450 enzyme of Oryza sativa 97C ABB47954 interest or a database comprising cytochrome P450 The start methionine (“ M ” ) may be present or absent from these sequences. sequences to identify the heme axial ligand . Such analyses * See , M . Z . Lv et at. , Plant Cell Physiol. , 53 ( 6 ) : 987 - 1002 ( 2012 ) . can be performed using the BLAST programs ( see , e . g . , Altschul et al. , J Mol Biol. 215 ( 3 ) :403 - 10 ( 1990 ) ) . Software In certain embodiments , the present invention provides for performing BLAST analyses publicly available through amino acid substitutions that efficiently remove monooxy the National Center for Biotechnology Information (http : / / genation chemistry from cytochrome P450 enzymes . This ncbi. nlm . nih . gov ) . BLASTP is used for amino acid system permits selective enzyme- driven cyclopropanation sequences . chemistry without competing side reactions mediated by Exemplary parameters for performing amino acid native P450 catalysis . The invention also provides P450 sequence alignments to identify the heme axial ligand in a mediated catalysis that is competent for cyclopropanation P450 enzyme of interest using the BLASTP algorithm chemistry but not able to carry out traditional P450 -medi - 20 include E value = 10 , word size = 3 , Matrix = Blosum62 , Gap ated monooxygenation reactions as 'orthogonal P450 opening = 11 , gap extension = 1, and conditional composi catalysis and respective enzyme variants as ' orthogonal tional score matrix adjustment. Those skilled in the art will P450s. In some instances , orthogonal P450 variants com know what modifications can be made to the above param prise a single amino acid mutation at the axial position of the eters , e . g . , to either increase or decrease the stringency of the heme coordination site ( e . g . , a C400S mutation in the P450 25 comparison and/ or to determine the relatedness of two or BM3 enzyme) that alters the proximal heme coordination more sequences . environment. Accordingly, the present invention also pro In preferred embodiments , the cytochrome P450 enzyme vides P450 variants that contain an axial heme mutation in is a cytochrome P450 BM3 enzyme or a variant, homolog , combination with one or more additional mutations described herein to provide orthogonal P450 variants that 30 or fragment thereof. The bacterial cytochrome P450 BM3 show enriched diastereoselective and / or enantioselective from Bacillus megaterium is a water soluble , long - chain product distributions. The present invention further provides fatty acid monooxygenase . The native P450 BM3 protein is a compatible reducing agent for orthogonal P450 cyclopro comprised of a single polypeptide chain of 1048 amino acids panation catalysis that includes, but is not limited to , NAD and can be divided into 2 functional subdomains (see , L . O . ( P )H or sodium dithionite . Narhi et al ., J Biol. Chem . 261, 7160 ( 1986 ) ) . An N -terminal In particular embodiments , the cytochrome P450 enzyme domain , amino acid residues 1 -472 , contains the heme is one of the P450 enzymes or enzyme classes set forth in bound active site and is the location for monoxygenation Table 2 or 3 . In some embodiments , the cytochrome P450 catalysis . The remaining C - terminal amino acids encompass enzyme is a variant or homolog of one of the P450 enzymes a reductase domain that provides the necessary electron or enzyme classes set forth in Table 2 or 3 . In preferred 40 equivalents from NADPH to reduce the heme cofactor and embodiments , the P450 enzyme variant comprises a muta - drive catalysis . The presence of a fused reductase domain in tion at the conserved cysteine (Cys or C ) residue of the P450 BM3 creates a self -sufficient monooxygenase , obviat corresponding wild - type sequence that serves as the heme ing the need for exogenous accessory proteins for oxygen axial ligand to which the iron in protoporphyrin IX is activation ( see , id . ) . It has been shown that the N - terminal attached . As non - limiting examples , axial mutants of any of 45 heme domain can be isolated as an individual, well - folded , the P450 enzymes set forth in Table 2 or 3 can comprise a soluble protein that retains activity in the presence of mutation at the axial position (“ AxX ” ) of the heme coordi- hydrogen peroxide as a terminal oxidant under appropriate nation site , wherein “ X ” is selected from Ala , Asp , Arg , Asn , conditions ( P . C . Cirino et al. , Angew . Chem ., Int. Ed . 42 , Glu , Gln , Gly , His , Ile , Lys, Leu , Met, Phe , Pro , Ser, Thr, 3299 (2003 ) ). Trp , Tyr, and Val. 50 In certain instances , the cytochrome P450 BM3 enzyme In certain embodiments, the conserved cysteine residue in comprises or consists of the amino acid sequence set forth in a cytochrome P450 enzyme of interest that serves as the SEQ ID NO : 1 . In certain other instances , the cytochrome heme axial ligand and is attached to the iron in protopor P450 BM3 enzyme is a natural variant thereof as described , phyrin IX can be identified by locating the segment of the e .g ., in J . Y . Kang et al. , AMB Express 1 : 1 (2011 ) , wherein DNA sequence in the corresponding cytochrome P450 gene 55 the natural variants are divergent in amino acid sequence which encodes the conserved cysteine residue . In some from the wild - type cytochrome P450 BM3 enzyme sequence instances, this DNA segment is identified through detailed (SEQ ID NO : 1 ) by up to about 5 % ( e . g ., SEQ ID NOS : 2 mutagenesis studies in a conserved region of the protein 11 ) . ( see , e . g . , Shimizu et al ., Biochemistry 27 , 4138 -4141 , In particular embodiments , the P450 BM3 enzyme variant 1988 ) . In other instances , the conserved cysteine is identified 60 comprises or consists of the heme domain of the wild - type through crystallographic study ( see , e . g . , Poulos et al ., J . P450 BM3 enzyme sequence ( e . g . , amino acids 1 - 463 of Mol . Biol 195 :687 -700 , 1987 ) . SEQ ID NO : 1 ) and optionally at least one mutation as In situations where detailed mutagenesis studies and described herein . In other embodiments , the P450 BM3 crystallographic data are not available for a cytochrome enzyme variant comprises or consists of a fragment of the P450 enzyme of interest, the axial ligand may be identified 65 heme domain of the wild - type P450 BM3 enzyme sequence through phylogenetic study . Due to the similarities in amino (SEQ ID NO : 1 ) , wherein the fragment is capable of acid sequence between P450 enzymes, standard protein carrying out the cyclopropanation reactions of the present US 10 , 208 , 322 B2 35 36 invention . In some instances , the fragment includes the a C400X mutation ( e. g ., C400S ) in SEQ ID NO : 1 or a heme heme axial ligand and at least one, two , three , four, or five domain or fragment thereof. In other instances , the P450 of the active site residues . BM3 enzyme variant comprises a T438 mutation such as In certain embodiments , the P450 BM3 enzyme variant T438A , T438S , or T438P in a BM3- CIS backbone alone or comprises a mutation at the axial position ( “ AxX ” ) of the 5 in combination with a C400X mutation ( e . g . , C400S ) in SEQ heme coordination site , wherein “ X ” is selected from Ala , ID NO : 1 ( i. e ., " BM3- CIS - T438S - AxX ” ) . In yet other Asp , Arg , Asn , Glu , Gin , Gly , His , Ile, Lys, Leu , Met, Phe , instances , the P450 BM3 enzyme variant comprises or Pro , Ser , Thr, Trp , Tyr, and Val. The conserved cysteine (Cys consists of the heme domain of the BM3 - CIS enzyme or C ) residue in the wild -type P450 BM3 enzyme is located sequence or a fragment thereof in combination with a T438 at position 400 in SEQ ID NO : 1 . As used herein , the terms 10 mutation and an “ AxX ” mutation ( e . g ., " BM3- CIS - T438S “ AxX ” and “ C400X ” refer to the presence of an amino acid AxX heme” ) . substitution “ X ” located at the axial position (i . e ., residue In other embodiments , the P450 BM3 enzyme variant 400 ) of the wild - type P450 BM3 enzyme ( i . e . , SEQ ID further comprises from one to five ( e . g ., one , two , three , NO : 1 ) . In some instances , X is Ser ( S ). In other instances, X four, or five ) active site alanine substitutions in the active is Ala ( A ) , Asp ( D ) , His ( H ) , Lys ( K ) , Asn ( N ) , Met ( M ), Thr 15 site of SEQ ID NO : 1 . In certain instances , the active site ( T ) , or Tyr ( Y ) . In some embodiments , the P450 BM3 alanine substitutions are selected from the group consisting enzyme variant comprises or consists of the heme domain of of 175A , M177A , L181A , 1263A , L437A , and a combina the wild -type P450 BM3 enzyme sequence (e . g. , amino tion thereof. acids 1- 463 of SEQ ID NO : 1 ) or a fragment thereof and an In further embodiments , the P450 BM3 enzyme variant AxX mutation ( i . e ., “ WT- AxX heme" ) . 20 comprises at least one or more ( e. g ., at least about 2 , 3 , 4 , 5 , In other embodiments , the P450 BM3 enzyme variant 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , or comprises at least one or more (e . g. , at least two , three , four, 22 ) of the following amino acid substitutions in SEQ ID five , six , seven , eight, nine , ten , eleven , twelve , or all NO : 1 : R47C , L521, 158V , L75R , F81 ( e . g . , F81L , F81W ) , thirteen ) of the following amino acid substitutions in SEQ A 82 ( e . g . , A82S , A82F , A82G , A82T , etc . ), F87A , K941, ID NO : 1 : 178A , F87V , P142S , T1751, A184V , S226R , 25 194K , H10OR , S106R , F107L , A135S , F1621, A1971 , H236Q , E252G , T268A , A290V, L353V , 1366V , and E442K . F205C , N239H , R255S , S274T , L3241, A328V , V340M , and In certain instances , the P450 BM3 enzyme variant com - K434E . In particular embodiments, the P450 BM3 enzyme prises a T268A mutation alone or in combination with one variant comprises any one or a plurality of these mutations or more additional mutations such as a C400X mutation alone or in combination with one or more additional muta ( e . g . , C400S ) in SEQ ID NO : 1 . In other instances , the P450 30 tions such as those described above , e . g ., an “ AxX ” muta BM3 enzyme variant comprises all thirteen of these amino tion and / or at least one or more mutations including V78A , acid substitutions ( i . e . , V78A , F87V , P142S , T1751, A184V , F87V , P142S , T1751, A184V , S226R , H2360 , E252G , S226R , H236Q , E252G , T268A , A290V, L353V , 1366V , and T268A , A290V , L353V , 1366V , and E442K . E442K ; “ BM3 -CIS ” ) in combination with a C400X muta - Table 4 below provides non - limiting examples of tion ( e . g ., C400S ) in SEQ ID NO : 1 . In some instances, the 35 cytochrome P450 BM3 variants of the present invention . P450 BM3 enzyme variant comprises or consists of the Each P450 BM3 variant comprises one or more of the listed heme domain of the BM3- CIS enzyme sequence ( e . g ., mutations ( Variant Nos. 1 -31 ) , wherein a “ + ” indicates the amino acids 1 -463 of SEQ ID NO : 1 comprising all thirteen presence of that particular mutation (s ) in the variant. Any of of these amino acid substitutions ) or a fragment thereof and the variants listed in Table 4 can further comprise an 1263A an “ AxX ” mutation ( i . e . , “ BM3 - CIS - AxX heme” ) . 40 and / or an A328G mutation and/ or at least one , two, three , In some embodiments , the P450 BM3 enzyme variant four, or five of the following alanine substitutions , in any further comprises at least one or more ( e . g . , at least two , or combination , in the P450 BM3 enzyme active site : L75A , all three ) of the following amino acid substitutions in SEQ M 177A , L181A , 1263A , and L437A . In particular embodi ID NO : 1 : 1263A , A328G , and a T438 mutation . In certain m ents , the P450 BM3 variant comprises or consists of the instances, the T438 mutation is T438A , T438S , or T438P . In 45 heme domain of any one of Variant Nos . 1 -31 listed in Table some instances, the P450 BM3 enzyme variant comprises a 4 or a fragment thereof, wherein the fragment is capable of T438 mutation such as T438A , T438S , or T438P alone or in carrying out the cyclopropanation reactions of the present combination with one or more additional mutations such as invention . TABLE 4 Exemplary cytochrome P450 BM3 enzyme variants of the present invention . P450 BM3 variant Mutation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

C400X + + + + + T268A + + ++ + + F87V + + + + + 9 - 10A - TS + ++ + + T438Z + + + + P450BM3 variant Mutation 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

1 C400X + + + + + + + + + T268A + + + + + + + + F87V + + + + + + + + + + US 10 , 208 , 322 B2 37 38 TABLE 4 - continued Exemplary cytochrome P450 BM3 enzyme variants of the present invention . 9 - 10A - TS + + + + + + + + + T438Z + + + + + + + + + + + Mutations relative to the wild - type P450B amino acid sequence ( SEQD NO : 1 ) ; “ X ” is selected from Ala , Asp , Arg , Asn , Glu , Gin , Gly , His , Ile, Lys , Leu , Met, Phe , Pro , Ser, Thr, Trp , Tyr, and Val; “ Z ” is selected from Ala , Ser, and Pro ; “ 9 - 10A - TS ” includes the following amino acid substitutions in SEQD NO : 1 : V78A , P142S , T1751, A184V , S226R , H2360 , E252G , A290V , L353V , 1366V , and E442K . One skilled in the art will understand that any of the 10 687 -700 , 1987 ). In yet other instances, protein sequence mutations listed in Table 4 can be introduced into any alignment algorithms can be used to identify the conserved cytochrome P450 enzyme of interest by locating the seg ment of the DNA sequence in the corresponding cytochrome amino acid residue . As non - limiting examples, the use of P450 gene which encodes the conserved amino acid residue BLAST alignment with the P450 BM3 amino acid sequence as described above for identifying the conserved cysteine 15 as the query sequence to identify the heme axial ligand site residue in a cytochrome P450 enzyme of interest that serves and / or the equivalent T268 residue in other cytochrome as the heme axial ligand . In certain instances, this DNA P450 enzymes is illustrated in Examples 3 and 9 . segment is identified through detailed mutagenesis studies in Table 5A below provides non -limiting examples of pre a conserved region of the protein ( see , e . g ., Shimizu et al ., m ferred cytochrome P450 BM3 variants of the present inven Biochemistry 27 , 4138 -4141 , 1988 ) . In other instances, the - tion . Table 5B below provides non - limiting examples of conserved amino acid residue is identified through crystalcatal . preferred chimeric cytochrome P450 enzymes of the present lographic study ( see, e .g ., Poulos et al. , J . Mol. Biol 195 : invention . TABLE 5A Exemplary preferred cytochrome P450 BM3 enzyme variants of the invention . P450BM3 variants Mutations compared to wild -type P450BM3 ( SEQ ID NO : 1 ) P450BM3- T268A T268A P450BM3- T268A - C400H T268 A + C400H P411BM3 (ABC ) C400S P411BM3- T268A T268 A + C400S P450BM3- T268A -F87V T268A + F87V 9 - 10A TS V78A , P142S , T1751 , A184V, S226R , H236Q , E252G , A290V , L353V , 1366V, E442K 9 - 10A - TS -F87V 9 - 10A TS + F87V H2A10 9 - 10A TS + F87V, L75A , L181A , T268A H2A10 - C400S 9 - 10A TS + F87V , L75A , L181A , T268A , C400S H2A10 - C400H 9 - 10A TS + F87V , L75A , L181A , T268A , C400H H2A10 - C400M 9 - 10A TS + F87V , L75A , L181A , T268A , C400M H2- 5 - F10 9 - 10A TS + F87V , L75A , 1263A , T268A , L437A H2- 5 - F10 - C400S 9 - 10A TS + F87V , L75A , 1263A , T268A , L437A , C400S H2 - 5 - F10 - C400H 9 - 10A TS + F87V , L75A , 1263A , T268A , L437A , C400H H2 - 5 - F10 - C400M 9 - 10A TS + F87V , L75A , 1263A , T268A , L437A , C400M H2 - 4 - D4 9 - 10A TS + F87V , L75A , M177A , L181A , T268A , L437A H2 - 4 - D4 - C400S 9 - 10A TS + F87V , L75A , M177A , L181A , T268A , L437A , C400S H2 - 4 - D4 - C400H 9 - 10A TS + F87V , L75A , M177A , L181A , T268A , L437A , C400H H2 - 2 - A1 9 - 10A TS + F87A , L75A , L181A , L437A H2- 2 -A1 - C400S 9 - 10A TS + F87A , L75A , L181A , L437A , C400S H2 - 2 - A1 - C400H 9 - 10A TS + F87A , L75A , L181A , L437A , C400H H2- 8 -C7 9 - 10A TS + L75A , F87V , L181A H2- 5 - F10 - A75L 9 - 10A TS + F87V - 1263A - T268A - L437A CH F8 R470 , V78A , K941, P142S , T1751, A184V, F205C , S226R , H236Q , E252G , R255S , A290V, L353V , F81W , A82S , F87A , A197V BM3- CIS (P450 BM3- CIS ; C3C ) 9 - 10A TS + F87V , T268A BM3- CIS - 1263A BM3 - CIS + 1263A BM3- CIS - A328G BM3- CIS + A328G BM3- CIS - T438S BM3 - CIS + T438S BM3- CIS - C400S (P411 BM3- CIS ; BM3- CIS + C400S ABC - CIS ) BM3- CIS - C400D ( BM3- CIS - AxD ) BM3- CIS + C400D BM3- CIS - C400Y ( BM3- CIS - AxY) BM3- CIS + C400Y BM3- CIS - C400K ( BM3-CIS - AxK ) BM3 - CIS + C400K BM3- CIS - C400H ( BM3- CIS - AxH ) BM3 - CIS + C400H BM3- CIS - C400M (BM3 - CIS - AxM ) BM3 -CIS + C400M WT- AXA ( heme ) WT heme domain ( amino acids 1 - 463 of SEQ ID NO : 1 ) + C400A WT-AxD ( heme ) WT heme domain ( amino acids 1 -463 of SEQ ID NO : 1 ) + C400D WT-AxH (heme ) WT heme domain ( amino acids 1 - 463 of SEQ ID NO : 1 ) + C400H WT- AxK (heme ) WT heme domain ( amino acids 1 - 463 of SEQ ID NO : 1 ) + C400K US 10 , 208 , 322 B2 39 40 TABLE 5A - continued Exemplary preferred cytochrome P450 BM3 enzyme variants of the invention . P450BM3 variants Mutations compared to wild - type P450BM3 (SEQ ID NO : 1 ) WT- AxM (heme ) WT heme domain ( amino acids 1- 463 of SEQ ID NO : 1) + C400M WT- AxN (heme ) WT heme domain (amino acids 1- 463 of SEQ ID NO : 1) + C400N WT-AxY ( heme ) WT heme domain ( amino acids 1 -463 of SEQ ID NO : 1) + C400Y BM3- CIS - T4385 - AXA BM3- CIS - T438S + C400A BM3- CIS - T4385 - AxD BM3- CIS - T438S + C400D BM3- CIS - T4385 - AxM BM3- CIS - T4385 + C400M BM3 -CIS - T4385 - AXY BM3- CIS - T438S + C400Y BM3- CIS - T4385 - AxT BM3-CIS - T438S + C400T 7 - 11D R47C , V78A , K941, P142S , T1751, A184V , F205C , 5226R , H236Q , E252G , R255S , A290V , L353V , A82F, A328V 7 - 11D - C400S R470 , V78A , K941, P142S , T1751, A184V, F205C , 5226R , H236Q , E252G , R2555 , A290V , L353V, A82F , A328V , C400S 12- 10C R47C , V78A , A82G , F87V , K941, P142S , T1751, A184V, F205C , 5226R , H236Q , E252G , R255S , A290V , A328V , L353V 22A3 L521, 158V , L75R , F87A , H100R , 5106R , F107L , A135S , F1621, A184V , N239H , 5274T , L3241, V340M , 1366V , K434E Man1 V78A , K941, P142S , T1751, A184V, F205C , 5226R , H236Q , E252G , R2555 , A290V , L353V , F81L , A82T, F87A , 194K

TABLE 5B 25 the background of wild -type P450 BM3. In certain embodi ments , trans - preferential mutations in combination with Exemplary preferred chimeric cytochrome additional mutations such as V78A , P142S , T1751, A184V , P450 enzymes of the invention . S226R , H236Q , E252G , A290V , L353V , 1366V, E442K , and Chimeric P450s Heme domain block sequence SEQ ID NO F87V ( from 9 - 10 - A - TS ) are also tolerated when in the 30 presence of additional mutations including , but not limited C2G9 22223132 X7 22312333 to , 1263A , L437A , L181A and /or L75A . These mutations are X7 - 12 12112333 isolated to the heme domain of P450 BM3 and are located C2E6 11113311 in various regions of the heme domain structure including X7 - 9 32312333 the active site and periphery . C2B12 32313233 TSP234 22313333 ???? 35 In certain embodiments , the present invention also pro VIUENvides P450 variants that catalyze enantioselective cyclopro panation with enantiomeric excess values of at least 30 % In particular embodiments , cytochrome P450 BM3 vari - ( comparable with wild - type P450 BM3) , but more prefer ants with at least one or more amino acid mutations such as , ably at least 80 % , and even more preferably at least > 95 % e . g . , C400X ( AxX ) , BM3- CIS , T438 , and / or T268A amino 40 for preferred product diastereomers. acid substitutions catalyze cyclopropanation reactions effi- In other aspects , the present invention provides chimeric ciently , displaying increased total turnover numbers and heme enzymes such as , e . g ., chimeric P450 proteins com demonstrating highly regio - and / or enantioselective product prised of recombined sequences from P450 BM3 and at least formation compared to the wild -type enzyme. one , two , or more distantly related P450 enzymes from As a non - limiting example , the cytochrome P450 BM3 45 Bacillus subtillis or any other organism that are competent variants of the present invention are cis - selective catalysts cyclopropanation catalysts using similar conditions to wild that demonstrate diastereomeric ratios at least comparable to type P450 BM3 and highly active P450 BM3 variants . As a wild -type P450 BM3, e . g ., at least 37 :63 cis : trans, at least non - limiting example , site - directed recombination of three 50 :50 cis : trans , at least 60 : 40 cis : trans, or at least 95 :5 bacterial cytochrome P450s can be performed with sequence cis : trans . Particular mutations for improving cis - selective 50 crossover sites selected to minimize the number of disrupted catalysis include at least one mutation comprising T268A , contacts within the protein structure . In some embodiments , C400X , and T438S , but preferably one, two , or all three of seven crossover sites can be chosen , resulting in eight these mutations in combination with additional mutations sequence blocks. One skilled in the art will understand that comprising V78A , P142S , T1751, A184V , S226R , H236Q , the number of crossover sites can be chosen to produce the E252G , A290V , L353V , 1366V , E442K , and F87V derived 55 desired number of sequence blocks, e. g ., 1 , 2 , 3, 4 , 5 , 6 , 7 , from P450 BM3 variant 9 - 10A - TS . These mutations are 8, or 9 crossover sites for 2 , 3 , 4 , 5 , 6 , 7, 8 , 9, or 10 sequence isolated to the heme domain of P450 BM3 and are located blocks, respectively . In other embodiments , the numbering in various regions of the heme domain structure including used for the chimeric P450 refers to the identity of the parent the active site and periphery . sequence at each block . For example, “ 12312312 ” refers to As another non - limiting example, the cytochrome P450 60 a sequence containing block 1 from P450 # 1 , block 2 from BM3 variants of the present invention are trans - selective P450 # 2 , block 3 from P450 # 3 , block 4 from P450 # 1 , block catalysts that demonstrate diastereomeric ratios at least 5 from P450 # 2 , and so on . A chimeric library useful for comparable to wild - type P450 BM3, e . g ., at least 37 :63 generating the chimeric heme enzymes of the invention can cis : trans, at least 20 :80 cis : trans , or at least 1 : 99 cis : trans . be constructed as described in , e . g ., Otey et al . , PLOS Particular mutations for improving trans -selective catalysis 65 Biology, 4 (5 ): e112 ( 2006 ), following the SISDC method include at least one mutation comprising including T268A ( see , Hiraga et al. , J . Mol. Biol. , 330 :287 - 96 (2003 ) ) using and C400X , but preferably one or both of these mutations in the type IIb restriction endonuclease BsaXI, ligating the US 10 , 208 , 322 B2 42 full- length library into the pCWori vector and transforming 900 , 950 , 1000 , 1500 , 2000 , 2500 , 3000 , 3500 , 4000 , 4500 , into the catalase - deficient E . coli strain SN0037 ( see , Naka - 5000 , 5500 , 6000 , 6500 , 7000 , 7500 , 8000 , 8500 , 9000 , gawa et al. , Biosci. Biotechnol. Biochem . , 60 :415 -420 9500 , 10 , 000 , 15 , 000 , 20 ,000 , 25 ,000 , 30 ,000 , 35 , 000 , ( 1996 ) ) ; the disclosures of these references are hereby 40 , 000 , 45 , 000 , 50 ,000 , 55 , 000 , 60 ,000 , 65 , 000 , 70 ,000 , incorporated by reference in their entirety for all purposes . 5 75 ,000 , 80 ,000 , 85 ,000 , 90 ,000 , 95 ,000 , 100 ,000 , or more. As a non - limiting example , chimeric P450 proteins com In certain embodiments , the variant or chimeric heme prising recombined sequences or blocks of amino acids from enzymes of the present invention have higher TTNs com CYP102A1 (Accession No . J04832 ), CYP102A2 ( Acces- pared to the wild - type sequences. In some instances, the sion No . CAB12544 ) , and CYP102A3 (Accession No. variant or chimeric heme enzymes have TTNs greater than U93874 ) can be constructed . In certain instances , the 10 about 100 ( e . g . , at least about 100 , 150 , 200 , 250 , 300 , 325 , CYP102A1 parent sequence is assigned “ 1 ” , the CYP102A2 350 , 400 , 450 , 500 , or more ) in carrying out in vitro parent sequence is assigned “ 2 ” , and the CYP102A3 is cyclopropanation reactions. In other instances , the variant or parent sequence assigned “ 3 ” . In some instances , each chimeric heme enzymes have TTNs greater than about 1000 parent sequence is divided into eight sequence blocks con - ( e . g ., at least about 1000 , 2500 , 5000 , 10 ,000 , 25 ,000 , taining the following amino acids ( aa ) : block 1 : aa 1 -64 ; 15 50 , 000 , 75 , 000 , 100 , 000 , or more) in carrying out in vivo block 2 : aa 65 - 122 ; block 3 : aa 123 - 166 ; block 4 : aa whole cell cyclopropanation reactions . 167 - 216 ; block 5 : aa 217 -268 ; block 6 : aa 269- 328 ; block 7 : When whole cells expressing a heme enzyme are used to aa 329 -404 ; and block 8 : aa 405 - end . Thus, in this example, carry out a cyclopropanation reaction , the turnover can be there are eight blocks of amino acids and three fragments are expressed as the amount of substrate that is converted to possible at each block . For instance , “ 12312312 ” refers to a 20 product by a given amount of cellular material. In general, chimeric P450 protein of the invention containing block 1 in vivo cyclopropanation reactions exhibit turnovers from at - 1 ( aa 1 -64 ) from CYP102A1, block 2 ( aa 65 - 122 ) from least about 0 . 01 to at least about 10 mmol gedwcdw - ? , wherein CYP102A2, block 3 ( aa 123 - 166 ) from CYP102A3, block 4 Scdw is the mass of cell dry weight in grams. For example , ( aa 167 - 216 ) from CYP102A1, block 5 ( aa 217 - 268 ) from the turnover can be from about 0 . 1 to about 10 mmol gow ! , CYP102A2 , and so on . See , e . g . , Otey et al ., PLOS Biology , 25 or from about 1 to about 10 mmol gedw ? , or from about 5 4 ( 5 ) :e112 ( 2006 ) . Non - limiting examples of chimeric P450 to about 10 mmol. gcdw - ? , or from about 0 .01 to about 1 proteins include those set forth in Table 5B (C209 , X7, mmol. ge ww - 1 , or from about 0 .01 to about 0 . 1 mmol. gcdw , X7 -12 , C2E6 , X7- 9 , C2B12, TSP234 ) and Table 13 . In some or from about 0 . 1 to about 1 mmol. gcdw - 1, or greater than 1 embodiments, the chimeric heme enzymes of the invention mmol. gcdww - + . The turnover can be about 0 . 01 , 0 . 015 , 0 . 02 , can comprise at least one or more of the mutations described 30 0 . 025 , 0 . 03 , 0 .035 , 0 . 04 , 0 .045 , 0 . 05 , 0 . 055 , 0 .06 , 0 . 065 , herein . 0 .07 , 0 .075 , 0 .08 , 0 .085 , 0 .09 , 0 .095 , 0 . 1 , 0 . 15 , 0 . 2 , 0 . 25 , 0 . 3 , In some embodiments, the present invention provides the 0 . 35 , 0 . 4 , 0 .45 , 0 . 5 , 0 . 55 , 0 .6 , 0 .65 , 0 . 7 , 0 . 75 , 0 . 8 , 0 . 85 , 0 . 9 , incorporation of homologous or analogous mutations to 0 .95 , 1 .0 , 1. 5 , 2 .0 , 2 .5 , 3. 0 , 3 .5 , 4 .0 , 4 . 5 , 5 .0 , 5 .5 , 6 . 0 , 6 .5 , C400X ( AxX ) and / or T268A in other cytochrome P450 7 . 0 , 7 . 5 , 8 . 0 , 8 . 5 , 9 . 0 , 9 . 5 , or about 10 mmol. gcdw - ? . enzymes and heme enzymes in order to impart or enhance 35 When whole cells expressing a heme enzyme are used to cyclopropanation activity . carry out a cyclopropanation reaction , the activity can fur As non - limiting examples, the cytochrome P450 can be a ther be expressed as a specific productivity , e . g ., concentra variant of CYP101A1 (SEQ ID NO : 25 ) comprising a tion of product formed by a given concentration of cellular C357X ( e . g . , C357S ) mutation , a T252A mutation , or a material per unit time, e . g ., in g / L of product per g / L of combination of C357X ( e . g . , C357S ) and T252A mutations, 40 cellular material per hour ( g gew - ? h - 1 ) . In general, in vivo wherein “ X ” is any amino acid other than Cys , or the cyclopropanation reactions exhibit specific productivities cytochrome P450 can be a variant of CYP2B4 (SEQ ID from at least about 0 . 01 to at least about 0 . 5 g gedw - ? h - , NO : 28 ) comprising a C436X ( e. g ., C436S ) mutation , a wherein Scdw is the mass of cell dry weight in grams. For T302A mutation , or a combination of C436X (e . g ., C436S ) example , the specific productivity can be from about 0 .01 to and T302A mutations , wherein “ X ” is any amino acid other 45 about 0 . 1 g gew - ? h - ? , or from about 0 . 1 to about 0 . 5 g than Cys , or the cytochrome P450 can be a variant of gew - 1 h - 1 , or greater than 0 . 5 g gew - 2h - 1 . The specific CYP2D7 ( SEQ ID NO : 26 ) comprising a C461X ( e . g ., Scdwproductivity can be about 0 .01 , 0 Podw.015 , 0 .02 , 0 . 025 , 0 .03 , C461S ) mutation , wherein “ X ” is any amino acid other than 0 .035 , 0 . 04 , 0 . 045, 0 .05 , 0 . 055 , 0 . 06 , 0 .065 , 0 . 07 , 0 .075 , Cys, or the cytochrome P450 can be a variant of P450C27 0 .08 , 0 .085 , 0 . 09 , 0 .095 , 0 .1 , 0 . 15 , 0 .2 , 0 .25 , 0 . 3 , 0 .35 , 0 .4 , (SEQ ID NO :27 ) comprising a C478X ( e . g ., C478S ) muta - 50 0 . 45 , or about 0 .5 g Scdw - ? h - . tion , wherein “ X ” is any amino acid other than Cys . In certain embodiments ,mutations can be introduced into An enzyme' s total turnover number ( or TTN ) refers to the the target gene using standard cloning techniques ( e . g . , maximum number of molecules of a substrate that the site - directed mutagenesis ) or by gene synthesis to produce enzyme can convert before becoming inactivated . In gen the heme enzymes ( e . g . , cytochrome P450 variants ) of the eral, the TTN for the heme enzymes of the invention range 55 present invention . The mutated gene can be expressed in a from about 1 to about 100 ,000 or higher . For example , the host cell ( e . g . , bacterial cell) using an expression vector TTN can be from about 1 to about 1 , 000 , or from about under the control of an inducible promoter or by means of 1 ,000 to about 10 , 000 , or from about 10 ,000 to about chromosomal integration under the control of a constitutive 100 ,000 , or from about 50 ,000 to about 100 ,000 , or at least promoter . Cyclopropanation activity can be screened in vivo about 100 , 000 . In particular embodiments , the TTN can be 60 or in vitro by following product formation by GC or HPLC from about 100 to about 10 ,000 , or from about 10 ,000 to as described herein . about 50 , 000 , or from about 5 , 000 to about 10 , 000 , or from The expression vector comprising a nucleic acid sequence about 1 ,000 to about 5 , 000 , or from about 100 to about that encodes a heme enzyme of the invention can be a viral 1 ,000 , or from about 250 to about 1 , 000 , or from about 100 vector, a plasmid , a phage, a phagemid , a cosmid , a fosmid , to about 500 , or at least about 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 65 a bacteriophage (e . g. , a bacteriophage P1 -derived vector 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , 150 , 200 , 250 , (PAC ) ) , a baculovirus vector , a yeast plasmid , or an artificial 300 , 350 , 400 , 450 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , 850 , chromosome ( e . g . , bacterial artificial chromosome (BAC ) , a US 10 , 208 , 322 B2 43 44 yeast artificial chromosome ( YAC ) , a mammalian artificial kidney (BHK ) cells, MDCK cells , NIH -3T3 fibroblast cells , chromosome (MAC ), and human artificial chromosome and any other immortalized cell line derived from a mam (HAC )) . Expression vectors can includesude chromosomal , non malian cell . chromosomal , and synthetic DNA sequences. Equivalent In certain embodiments , the present invention provides expression vectors to those described herein are known in 5 heme enzymes such as the P450 variants described herein that are active cyclopropanation catalysts inside living cells . the art and will be apparent to the ordinarily skilled artisan . As a non - limiting example , bacterial cells ( e . g ., E . coli) can The expression vector can include a nucleic acid sequence be used as whole cell catalysts for the in vivo cyclopropa encoding a heme enzyme that is operably linked to a nation reactions of the present invention . In some embodi promoter , wherein the promoter comprises a viral, bacterial , 10 ments , whole cell catalysts containing P450 enzymes with archaeal , fungal , insect , or mammalian promoter . In certain the equivalent C400X mutation are found to significantly embodiments , the promoter is a constitutive promoter . In enhance the total turnover number (TTN ) compared to in some embodiments , the promoter is an inducible promoter. vitro reactions using isolated P450 enzymes. In other embodiments , the promoter is a tissue- specific B . Compounds promoter or an environmentally regulated or a developmen - 15 . The methods of the invention can be used to provide a tally regulated promoter . number of cyclopropanation products . The cyclopropanation It is understood that affinity tags may be added to the N - products include several classes of compound including, but and /or C - terminus of a heme enzyme expressed using an not limited to , commodity and fine chemicals , flavors and expression vector to facilitate protein purification . Non scents , insecticides , and active ingredients in pharmaceutical limiting examples of affinity tags include metal binding tags 20 compositions. The cyclopropanation products can also serve such as His6 -tags (SEQ ID NO : 74 ) and other tags such as as starting materials or intermediates for the synthesis of glutathione S - transferase (GST ) . compounds belonging to these and other classes . Non - limiting expression vectors for use in bacterial host In some embodiments , the cyclopropanation product is a cells include pCWori, PET vectors such as pET22 (EMD compound according to formula Millipore) , PBR322 ( ATCC37017 ), PQETM vectors ( Qia - 25 gen ) , pBluescriptTM vectors (Stratagene ), pNH vectors , lambda -ZAP vectors (Stratagene ) ; ptrc99a , PKK223 - 3 , R ? R ? PDR540 , PRIT2T (Pharmacia ), pRSET, PCR - TOPO vectors , PET vectors, pSyn _ vectors , pChlamy _ 1 vectors (Life Tech nologies , Carlsbad , Calif. ), PGEM1 (Promega , Madison , 30 mR3 Wis .) , and PMAL (New England Biolabs, Ipswich , Mass. ). RERE Non - limiting examples of expression vectors for use in eukaryotic host cells include pXT1 , pSG5 (Stratagene ) , For compounds of Formula I, R ' is independently selected PSVK3, PBPV , PMSG , PSVLSV40 (Pharmacia 14 .), , 35 from H , optionally substituted C1- 18 alkyl, optionally sub pcDNA3 . 3 , pcDNA4 / TO , pcDNA6 / TR , PLenti6 / TR , PMT stituted C6- 10 aryl, optionally substituted 6 - to 10 -membered vectors (Life Technologies ) , PKLAC1 vectors , pKLAC2 heteroaryl, halo , cyano , C (O )ORla , C ( O )N (R ?) 2, C (O )RS , vectors (New England Biolabs ), pQETM vectors ( Qiagen ) , C ( O ) C ( O )OR , and Si( R8) z ; and R2 is independently BacPak baculoviral vectors , pAdeno - XTM adenoviral vectors selected from H , optionally substituted Cie alkyl, option ( Clontech ), and pBABE retroviral vectors . Any other vector 40 ally substituted C6- 10 aryl, optionally substituted 6 - to may be used as long as it is replicable and viable in the host 10 -membered heteroaryl, halo , cyano , C ( O ) OR ’ a , C ( O ) N cell. ( R ? ) , C ( O )RS , C ( O ) C ( O )ORS , and Si( R ) 3 . Rla and R2a are The host cell can be a bacterial cell, an archaeal cell, a independently selected from H , optionally substituted C1- 18 fungal cell , a yeast cell , an insect cell , or a mammalian cell. alkyl and - L - R . Suitable bacterial host cells include , but are not limited to , 45 When the moiety - L -RC is present, L is selected from a BL21 E . coli , DE3 strain E . coli, E . coli M15 , DH5a , bond , C ( R ') 2 - , and — NR2 C (R ') 2 - . Each RL is inde DH10B , HB101 , T7 Express Competent E . coli (NEB ) , B . pendently selected from H , C . alkyl, halo , CN , and subtilis cells , Pseudomonas fluorescens cells , and cyanobac SO2, and each RC is selected from optionally substituted terial cells such as Chlamydomonas reinhardtii cells and C610 aryl, optionally substituted 6 - to 10 -membered heter Synechococcus elongates cells . Non - limiting examples of 50 oraryl, and optionally substituted 6 - to 10 -membered het archaeal host cells include Pyrococcus furiosus, Metallo - erocyclyl . sphera sedula , Thermococcus litoralis, Methanobacterium For compounds of Formula I , R3, R4, RS, and R? are thermoautotrophicum , Methanococcus jannaschii , Pyrococ - independently selected from H , C1- 18 alkyl, C2- 18 alkenyl, cus abyssi , Sulfolobus solfataricus, Pyrococcus woesei, Sul- C2- 18 alkynyl, optionally substituted C6- 10 aryl, optionally folobus shibatae , and variants thereof. Fungal host cells 55 substituted C . - C . alkoxy , halo , hydroxy , cyano , include, but are not limited to , yeast cells from the genera C ( O )N ( R )2 , NR ' C (O )R® , C ( O )R® , C (O )OR® , and N (Rº ) 2. Saccharomyces ( e .g ., S . cerevisiae ), Pichia ( P. Pastoris ), Each R ' and R8 is independently selected from H , optionally Kluyveromyces ( e . g . , K . lactis ) , Hansenula and Yarrowia , substituted C1- 12 alkyl, optionally substituted C2- 12 alkenyl , and filamentous fungal cells from the genera Aspergillus, and optionally substituted C6- 10 aryl. Each R is indepen Trichoderma, and Myceliophthora . Suitable insect host cells 60 dently selected from H , optionally substituted C6- 10 aryl, and include , but are not limited to , Sf9 cells from Spodoptera optionally substituted 6 - to 10 -membered heteroaryl. Alter frugiperda , Sf21 cells from Spodoptera frugiperda , Hi- Five natively , two Rº moieties, together with the nitrogen atom to cells , BTI- TN -5B1 - 4 Trichophusia ni cells , and Schneider 2 which they are attached , can form 6 - to 18 -membered (S2 ) cells and Schneider 3 (S3 ) cells from Drosophila heterocyclyl. melanogaster . Non - limiting examples of mammalian host 65 Alternatively , R forms an optionally substituted 3 - to cells include HEK293 cells , HeLa cells , CHO cells , COS 18 -membered ring with R4 , or RS forms an optionally cells , Jurkat cells , NSO hybridoma cells , baby hamster substituted 3 to 18 -membered ring with R . R ’ or R4 can also US 10 ,208 , 322 B2 45 46 form a double bond with RS or R “ . R3 or R4 forms an -continued optionally substituted 5 - to 6 -membered ring with Rs or Rº. AS In some embodiments , the cyclopropanation product is a compound of formula I as described above , wherein R is C (O ) O -LR " ;R² is selected from H and optionally substituted C6 - 10 aryl; and R , R4, R " , and Rº are independently selected from H , optionally substituted C1- 6 alkyl, optionally substi tuted C2- 6 alkenyl, optionally substituted C2- 6 alkynyl, A6 optionally substituted C6- 10 aryl, and halo . Alternatively , R 10 can form an optionally substituted 3 to 18 -membered ring with R4, or R $ can form an optionally substituted 3 to 18 -membered ring with R . In such embodiments, the cyclo propanation product can be a pyrethroid or a pyrethroid precursor. 15 In general, pyrethroids are characterized by an ester core A7 having a structure according to Formula III :

(III ) 20

-

- - A8 0 - LRIC. - 25 -

-

-

R6 30 Formula III is presented above as a cyclopropyl carboxy late moiety (“ A ” ) esterified with an LRT moiety (“ B ” ) , with Rlc defined as for RC . The methods of the invention can be used to prepare pyrethroids and pyrethroid intermediates 35 having a variety of “ A ” moieties connected to any of a variety of “ B ” moieties. For example , the pyrethroids can have an “ A ” moiety selected from :

A10 Al WAT

A11 ?2 moters

A3 A12

Meo N M

A4 60 A13 At ACI At 65 At US 10, 208, 322 B2 47 48 - continued -continued A14 A23

A15 A24

A16 A25

A17 A26

C/ A18 30 A2

35

A19

A28 40 P1

A20 45

50 A29 A21

55

A22 CI CI 60 A30

-N

65 US 10, 208 , 322 B2 49 50 - continued -continued A31 B4

A32 B5 1 )

, and A33 15 B6

27 10 2 )

B7 For the A moieties listed above, Xl is selected from H , optionally substituted C1 - 6 alkyl , haloC1 - 6 alkyl, optionally , substituted C1- 6 alkoXy, haloCq - 6 alkyl, optionally substi tuted C1- 6 alkylthio , C1- 6 alkylsilyl , halo , and cyano . X2 is selected from H , chloro, and methyl. X is selected from H , methyl, halo , and CN . Each X4 is independently halo . Each X ' is independently selected from methyl and halo. X? is 30 B8 selected from halo , optionally substituted C1- 6 alkyl, and optionally substituted C1-6 alkoxy. X7 is selected from H , methyl, and halo . X® is selected from H , halo , and optionally substituted C - alkyl. Xº is selected from H , halo , optionally substituted Cl- , alkyl, C ( O ) O - (C1 - alkyl) , C (O ) - N (C1 - 6 35 B9 alkyl) 2 , and cyano . Z , Z², and Z are independently selected from H , halo , optionally substituted C1- 6 alkyl, and option ally substituted C6- 10 aryl, or Z ' and Z are taken together to form an optionally substituted 5 - to 6 -membered cycloalkyl or heterocyclyl group . The wavy line at the right of each 40 structure represents the point of connection between the A B10 moiety and a B moiety . The pyrethroids can have “ B ” moieties selected from :

B1 B11 na B2

B12 EZ B3

B13

z US 10, 208, 322 B2 51 ??"|52 - continued -continued B14 B21

B22

B15 1 ) ? B23

B16 15

B24

B17

B25

17

B18 B26

11

B19 ? B27 un

Puuuuu B28 50 naan B21 SO2

B29 B22 55 ??? B30

B23 60

B31

65 US 10 , 208 , 322 B2 53 54 -continued C1- 6 alkyl, furfuryl, C1- 6 alkoxy , (C2 - 6 alkenyl) oxy, C1- 12 B32 acyl, and halo . Y is selected from the group consisting of optionally substituted C1- 6 alkyl, optionally substituted C1- 6 alkoxy, and halo . The subscript m is an integer from 1 to 3 , 5 the subscript n is an integer from 1 to 5 , the subscript p is an integer from 1 to 4 , and the subscript q is an integer from B33 0 to 3. The wavy line at the left of each structure represents the point of connection between the B moiety and an A moiety . 10 The methods of the invention can be used to prepare pyrethroids having any A moiety joined to any B moiety . A given pyrethroid can have a structure selected from : A1- B1 , B34 A2 -B1 , A3- B1 , A4 -B1 , A5 - B1, A6 - B1 , A7 -B1 , A8- B1, (Y2 ) m A9 -B1 , A10 -B1 , A11 - B1, A12 -B1 , A13 - B1, A14 -B1 , A15 15 B1, A16 -B1 , A17 -B1 , A18 -B1 , A19 -B1 , A20 -B1 , A21 -B1 , A22 -B1 , A23 -B1 , A24 -B1 , A25 -B1 , A26 -B1 , A27 -B1 , A28 B1, A29- B1 , A30 -B1 , A31 -B1 , A32 -B1 , A33 -B1 , A1- B2 , B35 A2 - B2 , A3 -B2 , A4 -B2 , A5 -B2 , A6 - B2 , A7- B2 , A8 -B2 , A9 -B2 , A10 -B2 , A11 - B2 , A12 - B2 , A13 - B2 , A14 - B2 , A15 20 B2 , A16 - B2 , A17 - B2 , A18 - B2 , A19 - B2 , A20 - B2 , A21 - B2 , A22 -B2 , A23 - B2 , A24 -B2 , A25 - B2 , A26 -B2 , A27 - B2 , A28 B2, A29- B2 , A30 -B2 , A31 -B2 , A32 -B2 , A33 - B2 , A1- B3 , A2 -B3 , A3 - B3 , A4 -B3 , A5 - B3 , A6 -B3 , A7 - B3, A8 - B3 , A9 -B3 , A10 -B3 , A11 - B3 , A12 -B3 , A13 - B3, A14 - B3 , A15 B36 25 B3 , A16 - B3 , A17 -B3 , A18 -B3 , A19 -B3 , A20 - B3 , A21 - B3 , A22 -B3 , A23 - B3 , A24 - B3 , A25 -B3 , A26 -B3 , A27 -B3 , A28 B3 , A29 - B3 , A30 - B3 , A31 - B3 , A32 - B3 , A33 - B3 , A1- B4 , A2 - B4 , A3 -B4 , A4 -B4 , A5 - B4 , A6 -B4 , A7 -B4 , A8 - B4 , A9 -B4 , A10 - B4 , A11 -B4 , A12 -B4 , A13 - B4 , A14 - B4 , A15 30 B4 , A16 -B4 , A17 - B4 , A18 -B4 , A19 -B4 , A20 - B4 , A21 - B4 , A22 -B4 , A23 - B4 , A24 - B4 , A25 -B4 , A26 -B4 , A27 -B4 , A28 B37 B4 , A29 - B4 , A30 -B4 , A31 -B4 , A32 -B4 , A33 -B4 , A1 -B5 , A2 - B5 , A3 -B5 , A4 - B5 , A5- B5 , A6 - B5 , A7- B5 , A8 - B5 , (CH2 ) p , A9 -B5 , A10 -B5 , A11 - B5 , A12 -B5 , A13 -B5 , A14 - B5 , A15 35 B5 , A16 - B5 , A17 - B5 , A18 -B5 , A19 - B5 , A20 -B5 , A21 -B5 , A22 - B5 , A23 - B5 , A24 - B5 , A25 - B5 , A26 -B5 , A27 -B5 , A28 B38 B5 , A29 - B5 , A30 - B5 , A31- B5 , A32 - B5 , A33 - B5 , A1- B6 , A2 - B6 , A3- B6 , A4 - B6 , A5 - B6 , A6 - B6 , A7 - B6 , A8 - B6 , A9 -B6 , A10 - B6 , A11 - B6 , A12 - B6 , A13 - B6 , A14 - B6 , A15 40 B6 , A16 - B6 , A17 -B6 , A18 - B6 , A19 - B6 , A20 - B6 , A21- B6 , B39 A22 - B6 , A23 - B6 , A24 - B6 , A25 - B6 , A26 - B6 , A27 - B6 , A28 B6 , A29 - B6 , A30 - B6 , A31 - B6 , A32 - B6 , A33 - B6 , A1 - B7 . A2- B7 , A3 - B7 , A4 -B7 , A5 - B7 , A6 -B7 , A7 - B7 , A8 - B7 , A9 -B7 , A10 -B7 , A11 - B7 , A12 -B7 , A13 -B7 , A14 - B7, A15 Ys, and 45 B7, A16 -B7 , A17 -B7 , A18 -B7 , A19 - B7 , A20 -B7 , A21 -B7 , A22 - B7 , A23 - B7 , A24 - B7, A25 - B7 , A26 -B7 , A27 -B7 , A28 B7, A29 -B7 , A30 -B7 , A31 -B7 , A32 -B7 , A33 -B7 , A1- B8 , A2 - B8 , A3 - B8 , A4 -B8 , A5 - B8 , A6 -B8 , A7 - B8, A8 - B8 , B40 A9 - B8 , A10 - B8 , A11 - B8 , A12 - B8 , A13 - B8 , A14 - B8 , A15 50 B8 , A16 -B8 , A17 -B8 , A18 - B8 , A19 -B8 , A20 -B8 , A21 - B8 , A22 - B8 , A23 - B8 , A24 - B8 , A25 - B8 , A26 -B8 , A27 -B8 , A28 B8, A29 -B8 , A30 - B8, A31 - B8, A32 - B8, A33 -B8 , A1 -B9 , A2- B9 , A3 -B9 , A4- B9 , A5 - B9, A6 -B9 , A7- B9 , A8 -B9 , A9- B9 , A10 -B9 , A11 - B9 , A12 -B9 , A13 -B9 , A14 - B9 , A15 55 B9, A16 -B9 , A17 - B9 , A18 -B9 , A19 - B9 , A20 - B9 , A21 - B9 , A22 -B9 , A23 -B9 , A24 -B9 , A25 - B9, A26 -B9 , A27 -B9 , A28 B9, A29 - B9, A30 - B9, A31 - B9 , A32 - B9, A33 - B9, A1- B10 , For the B moieties listed above, each Y is independently A2 -B10 , A3 - B10 , A4 -B10 , A5 -B10 , A6 -B10 , A7 -B10 , selected from optionally substituted C1- 6 alkyl, optionally A8 -B10 , A9 -B10 , A10 -B10 , A11 -B10 , A12 -B10 , A13 -B10 , substituted C2- alkenyl, optionally substituted C2 -6 alkynyl, 60 A14 -B10 , A15 -B10 , A16 -B10 , A17 -B10 , A18 -B10 , A19 phenyl, and ( phenyl) C1 - 6 alkoxy . Each Y ' is independently B10 , A20 -B10 , A21 - 010 , A22 -B10 , A23 -B10 . A24 - B10 . selected from halo , optionally substituted C1- 6 alkyl, option - A25 -B10 , A26 -B10 , A27 -B10 , A28 -B10 , A29 - B10 , A30 ally substituted C2- 6 alkenyl, optionally substituted C1- 6 B10 , A31- B10 , A32- B10 , A33 -B10 , A1 -B11 , A2- B11, alkoxy , and nitro . Each Y is independently optionally A3 -B11 , A4 - B11, A5 - B11 , A6 -B11 , A7 -B11 , A8 - B11, substituted C1- 6 alkyl. Each Y4 is independently selected 65 A9 -B11 , A10 -B11 , A11 -B11 , A12 -B11 , A13 -B11 , A14 -B11 , from optionally substituted C1- 6 alkyl, optionally substituted A15 - B11 , A16 -B11 , A17 -B11 , A18 -B11 , A19 -B11 , A20 C2- 6 alkenyl, optionally substituted C2- 6 alkynyl, C6- 10 aryl - B11 , A21 - B11 , A22- B11 , A23 -B11 , A24 -B11 , A25- B11 , US 10 , 208, 322 B2 55 56 A26 -B11 , A27 -B11 , A28 -B11 , A29 - B11 , A30 -B11 , A31 A9- B23 , A10 -B23 , A11- B23, A12 -B23 , A13 -B23 , A14 B11 , A32 -B11 , A33 -B11 , A1 -B12 , A2 - B12 , A3 -B12 , B23 , A15 -B23 , A16 -B23 , A17 - B23 , A18 -B23 , A19 -B23 , A4 -B12 , A5 -B12 , A6 -B12 , A7- B12 , A8 -B12 , A9- B12 , A10 A20 -B23 , A21 -B23 , A22 -B23 , A23 -B23 , A24 -B23 , A25 B12 , A11 - B12 , A12 -B12 , A13 -B12 , A14 - B12 , A15 -B12 , B23 , A26 -B23 , A27 -B23 , A28 - B23 , A29 -B23 , A30 -B23 , A16 -B12 , A17 -B12 , A18 -B12 , A19 - B12 , A20 -B12 , A21 - 5 A31 -B23 , A32 -B23 , A33 -B23 , A1- B24 , A2 -B24 , A3 -B24 , B12 , A22 -B12 , A23 - B12 , A24 - B12 , A25 -B12 , A26 -B12 , A4 -B24 , A5 -B24 , A6 -B24 , A7 -B24 , A8 -B24 , A9 - B24 , A10 A27 -B12 , A28 -B12 , A29 -B12 , A30 -B12 , A31 -B12 , A32 B24 , A11 -B24 , A12 -B24 , A13 -B24 , A14 - B24 , A15 - B24 , B12 , A33 -B12 , A1- B13 , A2 - B13 , A3- B13 , A4 -B13 , A16 - B24 , A17 - B24, A18 - B24 , A19 -B24 , A20 -B24 , A21 A5 -B13 , A6 - B13 , A7 -B13 , A8 - B13 , A9 - B13 , A10 -B13 , B24 , A22 -B24 , A23 - B24 , A24 - B24 , A25 -B24 , A26 - B24 , A11 - B13 , A12 -B13 , A13 -B13 , A14 - B13 , A15 -B13 , A16 - 10 A27 -B24 , A28 -B24 , A29 - B24 , A30 -B24 , A31 - B24 , A32 B13 , A17 -B13 , A18 - B13 , A19 - B13 , A20 -B13 , A21 -B13 , B24 , A33 -B24 , A1 - B25 , A2 -B25 , A3- B25 , A4 - B25 , A22 - B13 , A23 -B13 , A24 -B13 , A25 - B13 , A26 - B13 , A27 A5 -B25 , A6 -B25 , A7 -B25 , A8 -B25 , A9 - B25 , A10 - B25 , B13 , A28 -B13 , A29 -B13 , A30 -B13 , A31 -B13 , A32- B13 , A11 -B25 , A12 - B25 , A13 - B25, A14 -B25 , A15 -B25 , A16 A33 - B13 , A1- B14 , A2- B14 , A3 -B14 , A4 -B14 , A5 -B14 , B25 , A17 - B25 , A18 - B25 , A19 -B25 , A20 - B25 , A21 - B25 , A6 -B14 , A7 -B14 , A8 -B14 , A9 -B14 , A10 - B14 , A11 -B14 , 15 A22 -B25 , A23 -B25 , A24 -B25 , A25 -B25 , A26 -B25 , A27 A12 -B14 , A13 -B14 , A14 -B14 , A15 -B14 , A16 -B14 , A17 B25 , A28 - B25 , A29 -B25 , A30 - B25 , A31- B25 , A32 -B25 , B14 , A18 -B14 , A19 -B14 , A20 -B14 , A21 -B14 , A22 -B14 , A33 - B25 , A1- B26 , A2 -B26 , A3 -B26 , A4 - B26 , A5 -B26 , A23 -B14 , A24 -B14 , A25 -B14 , A26 -B14 , A27 -B14 , A28 A6 - B26 , A7 - B26 , A8 - B26 , A9 - B26 , A10 - B26 , A11 - B26 , B14 , A29 -B14 , A30 - B14 , A31 - B14 , A32 -B14 , A33 -B14 , A12 -B26 , A13 -B26 , A14 -B26 , A15 - B26 , A16 - B26 , A17 A1- B15 , A2 -B15 , A3 -B15 , A4 -B15 , A5 -B15 , A6 -B15 , 20 B26 , A18 -B26 , A19 -B26 , A20 - B26 , A21 - B26 , A22 -B26 , A7 -B15 , A8- B15 , A9 -B15 , A10 -B15 , A11 -B15 , A12 -B15 , A23 -B26 , A24 -B26 , A25 -B26 , A26 -B26 , A27 -B26 , A28 A13 -B15 , A14 -B15 , A15 -B15 , A16 - B15 , A17 -B15 , A18 B26 , A29 - B26 , A30 -B26 , A31 - B26 , A32 - B26 , A33 -B26 . B15 , A19 -B15 , A20 - B15 , A21 -B15 , A22 -B15 , A23 -B15 , A1- B27 , A2- B27 , A3 -B27 , A4 -B27 , A5 - B27 , A6 -B27 , A24 - B15 , A25 -B15 , A26 -B15 , A27 -B15 , A28 - B15 , A29 A7 -B27 , A8 - B27 , A9 -B27 , A10 - B27 , A11 - B27 , A12 -B27 , B15 , A30 -B15 , A31 -B15 , A32 - B15 , A33 - B15 , A1 - B16 , 25 A13 - B27 , A14 -B27 , A15 -B27 , A16 - B27 , A17 - B27 , A18 A2 -B16 , A3- B16 , A4 -B16 , A5 -B16 , A6 - B16 , A7- B16 , B27 , A19 -B27 , A20 -B27 , A21 -B27 , A22 -B27 , A23 -B27 , AS - B16 , A9 - B16 , A10 - B16 , A11 - B16 , A12 - B16 , A13 -B16 , A24 - B27 , A25 - B27 , A26 - B27 , A27 - B27 , A28 -B27 , A29 A14 -B16 , A15 -B16 , A16 - B16 , A17 - B16 , A18 -B16 , A19 - B27 , A30 - B27 , A31 - B27 , A32 - B27 , A33 - B27 , A1 -B28 , B16 , A20 -B16 , A21 - B16 , A22 - B16 , A23 -B16 , A24 - B16 , A2 -B28 , A3- B28 , A4 - B28 , A5 -B28 , A6 - B28 , A7 - B28 , A25 - B16 , A26 -B16 , A27 -B16 , A28 - B16 , A29 -B16 , A30 - 30 A8 -B28 , A9 - B28 , A10 - B28 , A11 - B28 , A12 -B28 , A13 -B28 , B16 , A31 - B16 , A32 - B16 , A33 -B16 , A1- B17 , A2 - B17 , A14 -B28 , A15 -B28 , A16 -B28 , A17 -B28 , A18 -B28 , A19 A3 -B17 , A4 -B17 , A5 -B17 , A6 -B17 , A7 - B17 , A8 -B17 , B28, A20 -B28 , A21 -B28 , A22- B28 , A23 -B28 , A24 -B28 , A9 -B17 , A10 - B17 , A11 -B17 , A12 -B17 , A13 - B17 , A14 A25 -B28 , A26 -B28 , A27 -B28 , A28 -B28 , A29 -B28 , A30 B17 , A15 -B17 , A16 -B17 , A17 -B17 , A18 -B17 , A19 -B17 , B28 , A31 - B28 , A32- B28 , A33 -B28 , A1- B29 , A2 -B29 , A20 - B17 , A21- B17 , A22 -B17 , A23 -B17 , A24 -B17 , A25 - 35 A3- B29 , A4 -B29 , A5 - B29 , A6 -B29 , A7 - B29, A8 - B29 , B17 , A26 - B17 , A27 -B17 , A28 -B17 , A29 - B17 , A30 -B17 , A9- B29 , A10 -B29 , A11 -B29 , A12 - B29 , A13 -B29 , A14 A31 -B17 , A32 -B17 , A33 -B17 , A1- B18 , A2 -B18 , A3 -B18 , B29 , A15 -B29 , A16 - B29 , A17 -B29 , A18 -B29 , A19 - B29 , A4 -B18 , A5 -B18 , A6 -B18 , A7- B18 , A8 -B18 , A9- B18 , A10 A20 -B29 , A21 -B29 , A22 -B29 , A23 -B29 , A24 -B29 , A25 B18 , A11 -B18 , A12 - B18 , A13 - B18 , A14 - B18 , A15 -B18 , B29 , A26 - B29 , A27 -B29 , A28 -B29 , A29 - B29 , A30 -B29 , A16 -B18 , A17 - B18 , A18 - B18 , A19 - B18 , A20 - B18 , A21 - 40 A31 -B29 , A32 -B29 , A33 -B29 , A1- B30 , A2 - B30 , A3 -B30 , B18 , A22 -B18 , A23 - B18 , A24 -B18 , A25 -B18 , A26 -B18 , A4A4 -B30 , A5 -B30 , A6 -B30 , A7 -B30 , A8 - B30 , A9 - B30 , A10 A27 -B18 , A28 -B18 , A29 -B18 , A30 -B18 , A31 - B18 , A32 - B30 , A11 -B30 , A12 -B30 , A13 - B30 , A14 -B30 , A15 - B30 , B18 , A33 -B18 , A1- B19 , A2 -B19 , A3 -B19 , A4 -B19 , A16 -B30 , A17 -B30 , A18 -B30 , A19 - B30 , A20 - B30 , A21 A5 -B19 , A6 - B19 , A7- B19 , A8 - B19 , A9 - B19 , A10 -B19 , B30 , A22 - B30 , A23 -B30 , A24 -B30 , A25 - B30 , A26 -B30 , A11 - B19 , A12 -B19 , A13 - B19 , A14 - B19 , A15 -B19 , A16 - 45 A27 -B30 , A28 - B30 , A29 - B30 , A30 - B30 , A31 - B30 , A32 B19 , A17 -B19 , A18 - B19 , A19 -B19 , A20 -B19 , A21 -B19 , B30 , A33 - B30 , A1- B31, A2 - B31 , A3 -B31 , A4 - B31 , A22 -B19 , A23 -B19 , A24 -B19 , A25 - B19 , A26 -B19 , A27 - A5 -B31 , A6 -B31 , A7 - B31 , A8 - B31, A9 - B31 , A10 -B31 . B19 , A28 -B19 , A29 -B19 , A30 -B19 , A31 - B19 , A32 -B19 , 11 - B31 , A12 -B31 , A13 -B31 , A14 - B31 , A15 - B31 , A16 A33 - B19 , A1- B20 , A2- B20 , A3 -B20 , A4 - B20 , A5 -B20 , B31 , A17 -B31 , A18 -B31 , A19 - B31 , A20 -B31 , A21 - B31 , A6 -B20 , A7 -B20 , A8 - B20 , A9 -B20 , A10 -B20 , A11 -B20 , 50 A22 - B31, A23 - B31, A24 -B31 , A25 -B31 , A26 -B31 , A27 A12 - B20 , A13 - B20 , A14 -B20 , A15 - B20 , A16 - B20 , A17 B31 , A28 - B31, A29 -B31 , A30 - B31 , A31 -B31 , A32 -B31 , B20 , A18 -B20 , A19 -B20 , A20 - B20 , A21 - B20 , A22 -B20 , A33 -B31 , A1- B32 , A2 - B32 , A3 - B32 , A4 -B32 , A5 -B32 , A23 -B20 , A24 -B20 , A25 -B20 , A26 -B20 , A27 - B20 , A28 A6 - B32 , A7- B32 , A8 - B32 , A9 -B32 , A10 - B32 , A11 - B32 , B20 , A29 -B20 , A30 - B20 , A31 - B20 , A32 -B20 , A33 - B20 , A12 - B32 , A13 - B32 , A14 -B32 , A15 - B32 , A16 -B32 , A17 A1- B21 , A2 -B21 , A3 -B21 , A4 -B21 , A5 -B21 , A6 -B21 , 55 B32 , A18 - B32 , A19 -B32 , A20 - B32 , A21 - B32 , A22 -B32 , A7 -B21 , A8 -B21 , A9- B21 , A10 -B21 , A11 -B21 , A12 - B21, A23 - B32 , A24 -B32 , A25 -B32 , A26 - B32 , A27 - B32 , A28 A13 -B21 , A14 -B21 , A15 -B21 , A16 -B21 , A17 - B21 , A18 - B32 , A29 -B32 , A30 -B32 , A31 -B32 , A32 -B32 , A33 - B32, B21 , A19 -B21 , A20 - B21 , A21 - B21, A22 -B21 , A23 -B21 , A1- B33 , A2 -B33 , A3 -B33 , A4 -B33 , A5 - B33 , A6 -B33 , A24 -B21 , A25 -B21 , A26 - B21, A27 - B21 , A28 - B21, A29 A7 -B33 , A8 -B33 , A9- B33, A10 - B33 , A11 - B33, A12 - B33, B21 , A30 - B21 , A31 - B21 , A32 - B21 , A33 -B21 , A1 -B22 , 60 A13 - B33 , A14 - B33 , A15 -B33 , A16 - B33 , A17 - B33 , A18 A2- B22 , A3 -B22 , A4 - B22 , A5 -B22 , A6 - B22 , A7 -B22 , B33 , A19 -B33 , A20 - B33 , A21 -B33 , A22 -B33 , A23 - B33 , AS -B22 , A9 -B22 , A10 -B22 , A11 - B22 , A12 - B22 , A13 -B22 , A24 - B33 , A25 -B33 , A26 - B33 , A27 - B33 , A28 -B33 , A29 A14 -B22 , A15 -B22 , A16 -B22 , A17 -B22 , A18 -B22 , A19 . B33 , A30 -B33 , A31 - B33 , A32 - B33 , A33 - B33 , A1- B34 , B22 , A20 -B22 , A21 - B22 , A22 - B22 , A23 -B22 , A24 - B22 , A2 -B34 , A3- B34 , A4 - B34 , A5 -B34 , A6 - B34 , A7 - B34 , A25 -B22 , A26 -B22 , A27 -B22 , A28 -B22 , A29 -B22 , A30 - 65 A8 -B34 , A9 - B34 , A10 - B34 , A11 -B34 , A12 - B34 , A13 -B34 , B22 , A31 - B22 , A32 -B22 , A33 -B22 , A1- B23 , A2 -B23 , A14 - B34 , A15 -B34 , A16 -B34 , A17 -B34 , A18 -B34 , A19 A3- B23 , A4 -B23 , A5 - B23 , A6 -B23 , A7- B23 , A8 -B23 , B34 , A20 - B34 , A21 -B34 , A22 -B34 , A23 - B34 , A24 -B34 , US 10 , 208 , 322 B2 57 58 A25 - B34 , A26 -B34 , A27 -B34 , A28 - B34 , A29- B34, A30 In some embodiments , the methods of the invention can B34 , A31 - B34 , A32 - B34 , A33 - B34 , A1- B35 , A2 - B35 , be used to prepare pyrethroid intermediate compounds that A3- B35 , A4 - B35 , A5 -B35 , A6 -B35 , A7- B35 , A8 -B35 , can be converted to the pyrethroid compounds described A9 -B35 , A10 - B35 , A11 - B35 , A12 -B35 , A13 - B35 , A14 above. Alkyl esters of cyclopropanecarboxylic acid and B35 . A15 -B35 , A16 -B35 , A17 -B35 , A18 -B35 , A19 -B35 , 5 cyclopropanecarboxylic acid derivatives can be converted to A20 -B35 , A21 -B35 , A22 -B35 , A23 - B35 , A24 -B35 , A25 - a variety of pyrethroid compounds via reaction with appro B35 , A26 - B35 , A27 - B35 , A28 - B35 , A29 - B35 , A30 -B35 , priate alcohols . A31- B35 , A32 -B35 , A33 -B35 , A1- B36 , A2 -B36 , A3 - B36 , Accordingly , some embodiments of the invention provide A4 -B36 , A5 -B36 , A6 -B36 , A7- B36 , A8 - B36 , A9- B36 , A10 - methods as wherein the cyclopropanation product is a com B36 , A11 -B36 , A12 - B36 , A13 -B36 , A14 - B36 , A15 -B36 , " pound according to formula II : A16 - B36 , A17 - B36 , A18 - B36 , A19 -B36 , A20 -B36 , A21 B36 , A22 - B36 , A23 - B36 , A24 - B36 , A25 -B36 , A26 - B36 , A27 - B36 , A28 - B36 , A29 - B36 , A30 -B36 , A31 -B36 , A32 ( II ) B36 , A33 -B36 , A1- B37 , A2 -B37 , A3- B37 , A4 -B37 , A5 - B37 , A6 -B37 , A7 - B37 , A8 -B37 , A9 - B37 , A10 - B37 , R ? 40- Rla A11 - B37 , A12 -B37 , A13 - B37 , A14 -B37 , A15 - B37 , A16 mos B37 , A17 -B37 , A18 -B37 , A19 -B37 , A20 -B37 , A21 -B37 , A22 - B37 , A23 - B37 , A24 -B37 , A25 - B37 , A26 -B37 , A27 m3 B37 , A28 -B37 , A29 - B37 , A30 - B37 , A31- B37 , A32 -B37 , 20 RO R4 A33 - B37 , A1- B38 , A2- B38 , A3 -B38 , A4 - B38 , A5 - B38 , A6 -B38 , A7 -B38 , A8 - B38 , A9- B38 , A10 -B38 , A11 -B38 , A12 -B38 , A13 -B38 , A14 -B38 , A15 -B38 , A16 -B38 , A17 For compounds of formula II : Rla is C1- 6 alkyl and R² is B38, A18 - B38 , A19 -B38 , A20 -B38 , A21 - B38 , A22 - B38 , selected from H and optionally substituted C6 - 10 aryl. In A23 - B38 , A24 - B38 , A25 -B38 , A26 - B38 , A27 -B38 , A28 - 25 some embodiments , R2 is H . In some embodiments , the B38 , A29 - B38 , A30 - B38 , A31- B38 , A32 - B38 , A33 -B38 , compound of formula II is selected from : A1- B39 , A2 -B39 , A3 - B39, A4 -B39 , A5 -B39 , A6 - B39, A7 -B39 , A8 - B39 , A9 -B39 , A10 -B39 , A11 - B39, A12 -B39 , A13 - B39 , A14 -B39 , A15 -B39 , A16 - B39 , A17 - B39 , A18 B39 , A19 -B39 , A20 - B39 , A21- B39, A22 - B39 , A23 -B39 , 300 A24 - B39 , A25 - B39 , A26 - B39 , A27 - B39 , A28 - B39, A29 LORla LORla , B39 , A30 - B39, A31 - B39, A32 - B39 , and A33 - B39 . The A moiety is joined to the B moiety to form the ester bond as shown in Formula III above. A pyrethroid prepared according to the methods of the 35 invention can have , for example , a structure selected from : ORla LORla

0 - LRIC, OR la OR la mu

20 - LRIC,

OR la LORI ,

20 - LRI , and

OR la 20 - LRIC

Z ', Z2, and Z are independently selected from H , halo , JORla , LOR la optionally substituted C1- 6 alkyl, and optionally substituted > Co - 10 aryl. Z and Z can also be taken together to form an 65 optionally substituted 5 - to 6 -membered cycloalkyl or het erocyclyl group . US 10 , 208 , 322 B2 59 60 -continued -continued CI CI

Meo Orla , 012 LORla, Nam man COORla ,

LORla LORla 10

COORla,

- ORla

20 22 . COORla z2 MCOORla ,

OR la = COORla , and C1

LORla 22 COORla In such embodiments , Xl is selected from H , optionally substituted C1- 6 alkyl, haloC1- 6 alkyl, optionally substituted C1- 6 alkoxy, optionally substituted C1- 6 alkylthio , C1- 6 alkyl 35 silyl, halo , and cyano . X² is selected from H , chloro , and ORla LORIA methyl. X is selected from H , methyl, halo , and CN . Each X4 is independently halo . Each X is independently selected from methyl and halo . X “ is selected from halo , optionally Ci Ci substituted C1- 6 alkyl, and optionally substituted C1- 6 40 alkoxy. X7 is selected from H , methyl, and halo . XS is selected from H , halo , and optionally substituted C - , alkyl. COORla LORla X® is selected from H , halo , optionally substituted C1- 6 alkyl, C ( O ) O - ( C ) - , alkyl ) , C ( O ) - N ( C - alkyl) 2 , and cyano . Z ' , Z ?, and Z are independently selected from H , halo , option xl 45 ally substituted C1- 6 alkyl, and optionally substituted C6- 10 aryl. Z and Z2 can also be taken together to form an optionally substituted 5 - to 6 -membered cycloalkyl or het erocyclyl group ORla In some embodiments , the methods of the invention 50 include converting the cyclopropanation product according to formula II to a compound according to formula III : XI"

(III ) > > CORla, 55 RSO - LRI . MR3 60 RERE For compounds of formula III , L is selected from a bond , LOR , X6 COORla , _ C (R ) 2 - , and — NR ? — C (R ) 2 — . Each R ' is indepen 65 dently selected from H , CN , and — SO2. Rle is selected from optionally substituted C6 -10 aryl , optionally substituted 6 - to 10 -membered heteroraryl , and optionally substituted 6 US 10 , 208 , 322 B2 62 to 10 -membered heterocyclyl. In some embodiments , L in - continued the compounds of formula III is selected from a bond , CH2 - , - CH (CN ) — , and — N (SO2 ) CH2. In some embodiments , the moiety L - Rlc in the com ! pounds according to formula III has a structure selected from : mm

10

15

ZE 20

25

ZA n

30 Loom

pooEZ :35 Lound xoxon40 45 Prao . gegnmm 50

55

SU2 mu US 10 , 208 , 322 B2 63 64 - continued -continued

5

Meo cici became10 ( Y2 ) n ,

15

Los Lobao and (CH2 ) p 20 F3C Z BLOG otaoCICI 19 Y5 , and 25 wongofjasC1 30 In some embodiments , the compound of formula III is resmethrin . As for the pyrethroids discussed herein , a number of other y5 35 compounds can be synthesized via processes that include a cyclopropanation product. Such processes are generalized in In such embodiments , each Y is independently selected Scheme 1 showing the enzyme- catalyzed formation of a from optionally substituted C1- 6 alkyl, optionally substituted cyclopropanation product from an olefinic substrate and a C2- 6 alkenyl, optionally substituted C2- 6 alkynyl, phenyl, and diazo reagent , followed by chemical conversion of to a final (phenyl ) C , alkoxy . Each Y2 is independently selected from 40 product such as a pharmaceutical agent. Depending on the halo , optionally substituted C1- 6 alkyl, optionally substituted particular final product , the process can include conversion C2- 6 alkenyl, optionally substituted C1. 6 alkoxy, and nitro . of the cyclopropanation product to one or more synthetic Each Y ’ is independently optionally substituted C . , alkyl. intermediates prior to preparation of the final product. Non Each Y4 is independently selected from optionally substi - limiting examples of cyclopropanation products useful in tuted C . alkyl, optionally substituted C26 alkenyl, option - 45 such processes are summarized in Table 6 . ally substituted C2- 6 alkynyl, C6- 10 aryl- C , - alkyl, furfuryl, C1- 6 alkoxy , (C2 - 6 alkenyl) oxy, C1- 12 acyl, and halo . Y is selected from the group consisting of optionally substituted Scheme 1 C1- 6 alkyl, optionally substituted Cl- alkoxy , and halo . The subscript m is an integer from 1 to 3 , the subscript n is an 50 heme integer from 1 to 5 , the subscript p is an integer from 1 to R: R enzyme 4 , and the subscript q is an integer from 0 to 3 . The wavy line LR + at the left of each structure represents the point of connection N2 between the moiety - L -RC and the rest of the compound diazo according to formula III . olefinic reagent In some embodiments, the compound of formula III is substrate selected from : R ? R ?

step ( s ) R3 step ( s ) intermediate (s ) 60 2 RER? cyclopropanation product de final orto65 product US 10 ,208 , 322 B2 65 TABLE 6 Cyclopropanation for synthesis of intermediates en route to biologically active compounds. Cyclopropanation Olefinic Substrate Diazo Reagent Product/ Intermediate Final Product ÇONEt2 ÇONEt2 milnacipran

R = ORla , N (R7 ) 2 milnacipran

N2

ÇO R8 ÇORS milnacipran ; bicifidine ; 1 - ( 3 , 4 R dichlorophenyl) - 3 azabicyclo [3 . 1. 0 ] hexane R = ORla , N ( R ?) 2 ÇO R8 CONEt2 milnacipran ; bicifidine; 1 - ( 3 , 4 dichlorophenyl) - 3 azabicyclo [ 3 . 1 . 0 ] hexane N2 R = Orla, N (R )2 OR cilastain

R = Orla, N (R ?) 2 boceprevir

-ORS OR8 OR: ORS boceprevir

-OR : OR la TORS ORIO O boceprevir

PG OMe PG = protecting group NH US 10 ,208 , 322 B2 68 TABLE 6 - continued Cyclopropanation for synthesis of intermediates en route to biologically active compounds . Cyclopropanation Olefinic Substrate Diazo Reagent Productpole / Intermediatememeliste FinalFinal PProduct

1R ,2S fluorocyclopropyl amine , OR la sitafloxacin CO2Rla

TMS fluorocyclopropyl1R , 2S amine , sitafloxacin

TMS X7

anthoplalone , noranthoplone R N2 COEL R = ORI , N (R ]) 2 CN HH odanacatib (R72N ( R ]) 2ND

odanacatib

(R72N ( R ) 2N

ORS H montekulast (R7 ) 2N RS.

ORS montekulast (R3 ) 2ND

Orla Mex Me carene

COOME pyrethrin II

COOME MeOOC MeOOC US 10 ,208 , 322 B2 69 In some embodiments , the cyclopropanation product is a compound having a structure according to the formula :

0 - Rla OH . Pull NH2, H2N Roar 10 Et2NOC CONEt2 wherein Ria is optionally substituted C1- 6 alkyl, and Rs and Rº are independently selected from H , optionally substituted C1- 6 alkyl, optionally substituted C6- 10 aryl, C ( O ) N (R7 ) 2 , C (O )OR® and NR ' C ( O )RS . 15 In some embodiments , the cyclopropanation product has the structure selected from : NH COOEt, EtOOC 20 Et2NOC · CONET N1 Ph Ph

CH3, 25

Eto -NH2 Eto NH2 30 CH3, ' N

11 *# Eto - OR : 35 N

HO OH

40 NH2 ON NH • Eto — NH2 PC 45

Eto - ORS 50 ZI HNCN , In such embodiments , the methods of the invention can include converting the cyclopropanation product to a com - se pound selected from milcanipran , levomilnacipran , bicifa MeOS dine , and 1- ( 3 , 4 -dichlorophenyl ) - 3 - azabicyclo [3 . 1. 0 ] OH hexane . The methods of the invention can be used to prepare several different types of compounds having cyclopropane 60 functional groups. The compounds include , but are not limited to , pharmaceutical agents having chiral cyclopro pane moieties , pharmaceutical agents having achiral cyclo CI propane moieties, insecticides , plant hormones , flavors , HC scents , and fatty acids. 65 In some embodiments , the methods of the invention are used to prepare a compound selected from : US 10 , 208 , 322 B2 72 - continued -continued

OMe N NH NH ?? . ?á ANH - NH 10 >:11|

.lit

NH2, 15

Z Synthesis of prostratin , a protein kinase C activator , is shown as a non - limiting example in Scheme 2 . The pros tratin cyclopropane moiety can be installed by heme 20 enzyme- catalyzed intramolecular or intermolecular cyclo propanation reactions .

Scheme 2 ?? , 25

Ou 30 heme enzyme

H 35 O ' HO OH

40

45

H 50 O HO O HO - OH - ??

55

???

OH and heme 6060 S2 enzyme

HH

HO H?N 65 O -OH US 10 , 208 , 322 B2 73 -continued wherein Rla is selected from H and optionally substituted C .- C . alkyl; and each R7 and R8 is independently selected from H , optionally substituted C1- 12 alkyl, optionally sub stituted C2- 12 alkenyl, and optionally substituted C6- 10 aryl. 5 In some embodiments , the diazo reagent is selected from the group consisting of diazomethane , ethyl diazoacetate , and (trimethylsilyl ) diazomethane . In some embodiments , the diazo reagent is an a - diaz oester. In some embodiments , the diazo reagent has the 10 formula : ??? OH

Some embodiments of the invention provide a method as 15 described above , wherein the olefinic substrate is selected ORIG from the group consisting of an alkene , a cycloalkane , and an arylalkene . In some embodiments , the olefinic substrate is an arylalkene . In some embodiments , the arylalkene is a styrene. In some embodiments , the cyclopropanation product has In some embodiments, the styrene has the formula : 20 a formula selected from :

1 R 16 R . 25 R 10 ), COORla Po and R5 R6 R? is selected from the H , optionally substituted C . - C . alkyl, 30 optionally substituted C -Co alkoxy, C ( O ) N ( R ) , C ( O )OR® , COORla N (Rº ) 2 , halo , hydroxy , and cyano . R5 and R are indepen dently selected from H , optionally substituted C - , alkyl , and halo . R1° is selected from optionally substituted C . - C . alkyl, optionally substituted C . - C . alkoxy , halo , and haloalkyl, and (R9 ), the subscript r is an integer from 0 to 2 . In general, the diazo reagents useful in the methods of the One of skill in the art will appreciate that stereochemical invention have the structure : configuration of the cyclopropanation product will be deter 40 mined in part by the orientation of the diazo reagent with R ? R , respect to the position of an olefinic substrate such as styrene during the cyclopropanation step . For example , any sub stituent originating from the olefinic substrate can be posi tioned on the same side of the cyclopropyl ring as a 45 substituent origination from the diazo reagent . Cyclopropa wherein R and R2 are defined as for the cyclopropanation nation products having this arrangement are called “ cis ” products. Any diazoreagent can be added to the reaction as compounds or “ Z ” compounds . Any substituent originating a reagent itself, or the diazoreagent can be prepared in situ . from the olefinic substrate and any substituent originating In some embodiments , the diazo reagent is selected from from the diazo reagent can also be on opposite sides of the an a - diazoester , an a - diazoamide , an a - diazonitrile, an 50 cyclopropyl ring . Cyclopropanation products having this a -diazoketone , an a -diazoaldehyde , and an a -diazosilane . arrangement are called “ trans” compounds or “ E ” com In some embodiments , the diazo reagent has a formula pounds. An example of such arrangements is shown in the selected from : reaction scheme of FIG . 29 . As shown in FIG . 29 , two cis isomers and two trans 55 isomers can arise from the reaction of an olefinic substrate with a diazo reagent. The two cis isomers are enantiomers with respect to one another, in that the structures are 2 non -superimposable mirror images of each other . Similarly , N (R ), the two trans isomers are enantiomers . One of skill in the art N2 N2 N2Ñ 60 will appreciate that the absolute stereochemistry of a cyclo propranation product — that is , whether a given chiral center exhibits the right - handed “ R ” configuration or the left Si( R ) 3 handed “ S ” configuration will depend on factors including R $ Y R8, and the structures of the particular olefinic substrate and diazo N2 65 reagent used in the reaction , as well as the identity of the enzyme. This is also true for the relative stereochemistry that is , whether a cyclopropanation product exhibits a cis or US 10 ,208 , 322 B2 75 76 trans configuration as well as for the distribution of cyclo invention . In some embodiments , the invention provides any propanation product mixtures will also depend on such of the compounds illustrated in Table 6 , which compounds factors . are prepared according to the methods of the invention . The In general, cyclopropanation product mixtures have cis : invention can provide other compounds prepared according trans ratios ranging from about 1 : 99 to about 99 : 1 . The 5 to the methods described herein . cis : trans ratio can be , for example , from about 1 : 99 to about C . Reaction Conditions 1 : 75 , or from about 1 : 75 to about 1 : 50 , or from about 1 : 50 The methods of the invention include forming reaction to about 1 : 25 , or from about 99 : 1 to about 75 : 1 , or from mixtures that contain the heme enzymes described herein . about 75 : 1 to about 50 : 1 , or from about 50 : 1 to about 25 : 1 . The heme enzymes can be , for example , purified prior to The cis : trans ratio can be from about 1 : 80 to about 1 : 20 , or 10 addition to a reaction mixture or secreted by a cell present from about 1 :60 to about 1 :40 , or from about 80 : 1 to about in the reaction mixture . The reaction mixture can contain a 20 : 1 or from about 60 : 1 to about 40 : 1 . The cis : trans ratio can cell lysate including the enzyme , as well as other proteins be about 1 : 5 , 1 : 10 , 1 : 15 , 1 : 20 , 1 : 25 , 1 : 30 , 1 : 35 , 1 :40 , 1 :45 , and other cellular materials . Alternatively, a heme enzyme 1 : 50 , 1 : 55 , 1 :60 , 1 :65 , 1 : 70 , 1 :75 , 1 : 80 , 1 : 85 , 1 : 90 , or about can catalyze the reaction within a cell expressing the heme 1 : 95 . The cis : trans ratio can be about 5 : 1 , 10 : 1 , 15 : 1 , 20 : 1 , 15 enzyme. Any suitable amount of heme enzyme can be used 25 : 1 , 30 : 1 , 35 : 1 , 40 : 1 , 45 : 1 , 50 : 1 , 55 : 1 , 60 : 1 , 65 : 1 , 70 : 1 , in the methods of the invention . In general , cyclopropana 75 : 1 , 80 : 1 , 85 : 1 , 90 : 1 , or about 95 : 1 . tion reaction mixtures contain from about 0 .01 mol % to The distribution of a cyclopropanation product mixture about 10 mol % heme enzyme with respect to the diazo can be assessed in terms of the enantiomeric excess , or " % reagent and /or olefinic substrate . The reaction mixtures can ee , ” of the mixture . The enantiomeric excess refers to the 20 contain , for example , from about 0 . 01 mol % to about 0 . 1 difference in the mole fractions of two enantiomers in a mol % heme enzyme, or from about 0 . 1 mol % to about 1 mixture . Taking the reaction scheme in FIG . 29 as a non - mol % heme enzyme, or from about 1 mol % to about 10 mol limiting example , for instance , the enantiomeric excess of % heme enzyme. The reaction mixtures can contain from the “ E ” or trans ( R , R ) and ( S , S ) enantiomers can be calcu - about 0 . 05 mol % to about 5 mol % heme enzyme, or from lated using the formula : % eer = WRR - Xss / XR RtXssx 25 about 0 . 05 mol % to about 0 . 5 mol % heme enzyme. The 100 % , wherein x is themole fraction for a given enantiomer. reaction mixtures can contain about 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , The enantiomeric excess of the “ Z ” or cis enantiomers ( % 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , or about 1 mol % heme enzyme . een ) can be calculated in the same manner. The concentration of olefinic substrate and diazo reagent In general, cyclopropanantion product mixtures exhibit % are typically in the range of from about 100 uM to about 1 ee values ranging from about 1 % to about 99 % , or from 30 M . The concentration can be , for example , from about 100 about - 1 % to about - 99 % . The closer a given % ee value is uM to about 1 mM , or about from 1 mM to about 100 mm , to 99 % ( or - 99 % ), the purer the reaction mixture is . The % or from about 100 mM to about 500 mM , or from about 500 ee can be , for example , from about - 90 % to about 90 % , or mM to 1 M . The concentration can be from about 500 uM from about - 80 % to about 80 % , or from about - 70 % to to about 500 mM , 500 uM to about 50 mm , or from about about 70 % , or from about - 60 % to about 60 % , or from about 35 1 mM to about 50 mM , or from about 15 mM to about 45 - 40 % to about 40 % , or from about - 20 % to about 20 % . The mM , or from about 15 mM to about 30 mM . The concen % ee can be from about 1 % to about 99 % , or from about tration of olefinic substrate or diazo reagent can be, for 20 % to about 80 % , or from about 40 % to about60 % , or from example , about 100 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , or about 1 % to about 25 % , or from about 25 % to about 50 % , 900 uM . The concentration of olefinic substrate or diazo or from about 50 % to about 75 % . The % ee can be from 40 reagent can be about 1 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , about - 1 % to about - 99 % , or from about - 20 % to about 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , 150 , 200 , 250 , 300 , - 80 % , or from about - 40 % to about - 60 % , or from about 350 , 400 , 450 , or 500 mM . - 1 % to about - 25 % , or from about - 25 % to about – 50 % , or Reaction mixtures can contain additional reagents. As from about – 50 % to about - 75 % . The % ee can be about non - limiting examples , the reaction mixtures can contain - 99 % , - 95 % , - 90 % , - 85 % , - 80 % , - 75 % , - 70 % , - 65 % , 45 buffers ( e .g . , 2 - ( N -morpholino Jethanesulfonic acid (MES ) , - 60 % , - 55 % , - 50 % , - 45 % , - 40 % , - 35 % , - 30 % , - 25 % , 2 - [ 4 -( 2 -hydroxyethyl )piperazin - 1 - yl ] ethanesulfonic acid - 20 % , - 15 % , - 10 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , (HEPES ) , 3 -morpholinopropane - 1 - sulfonic acid (MOPS ), 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 2 - amino - 2 -hydroxymethyl - propane - 1 , 3 - diol ( TRIS ), potas 90 % , or about 95 % . Any of these values can be % ee , values sium phosphate , sodium phosphate , phosphate - buffered or % eez values . 50 saline , sodium citrate , sodium acetate , and sodium borate ) , Accordingly , some embodiments of the invention provide cosolvents (e .g . , dimethylsulfoxide , dimethylformamide , methods for producing a plurality of cyclopropanation prod ethanol, methanol, isopropanol , glycerol, tetrahydrofuran , ucts having a % eez of from about - 90 % to about 90 % . In acetone , acetonitrile , and acetic acid ), salts (e .g . , NaCl, KCI, some embodiments , the % ee , is at least 90 % . In some CaCl ,, and salts of Mn2 + and Mg2 + ) , denaturants ( e . g . , urea embodiments , the % eez is at least - 99 % . In some embodi - 55 and guandinium hydrochloride) , detergents ( e . g . , sodium ments , the % eeg is from about - 90 % to about 90 % . In some dodecylsulfate and Triton - X 100 ) , chelators (e . g . , ethylene embodiments, the % ee , is at least 90 % . In some embodi- glycol- bis ( 2 - aminoethylether ) - N , N , N ', N ' - tetraacetic acid ments , the % eer is at least - 99 % . (EGTA ) , 2 - { { 2 - [Bis ( carboxymethyl) amino ) ethyl } ( car In a related aspect , certain embodiments of the invention boxymethyl ) amino Jacetic acid (EDTA ) , and 1 , 2 -bis ( 0 provide cyclopropane - containing compounds according to 60 aminophenoxy ) ethane - N , N , N , N - tetraacetic acid any of Formulas I, II , III as described herein . The com - (BAPTA ) ), sugars (e . g. , glucose, sucrose, and the like ), and pounds are prepared using the methods of the invention . In reducing agents (e .g ., sodium dithionite , NADPH , dithio some embodiments , the invention provides a pyrethroid threitol (DTT ) , B -mercaptoethanol (BME ), and tris ( 2 - car prepared according to the methods of the invention . In some boxyethyl) phosphine ( TCEP ) ). Buffers , cosolvents, salts , embodiments , the invention provides milnacipran , levomil - 65 denaturants , detergents , chelators , sugars , and reducing nacipran , bicifadine , or 1 - ( 3 ,4 - dichlorophenyl) - 3 -azabicyclo agents can be used at any suitable concentration , which can [ 3 . 1 . 0 ]hexane prepared according to the methods of the be readily determined by one of skill in the art. In general , US 10 , 208 , 322 B2 77 78 buffers , cosolvents, salts, denaturants , detergents , chelators , particular enantiomer constituting 90 % of the total product sugars , and reducing agents , if present, are included in can be said to be 90 % enantioselective . A reaction producing reaction mixtures at concentrations ranging from about 1 uM a particular diastereomer constituting 30 % of the total to about 1 M . For example , a buffer , a cosolvent, a salt , a product, meanwhile , can be said to be 30 % diastereoselec denaturant, a detergent, a chelator , a sugar, or a reducing 5 tive . agent can be included in a reaction mixture at a concentra - In general, the methods of the invention include reactions tion of about 1 uM , or about 10 uM , or about 100 UM , or that are from about 1 % to about 99 % diastereoselective . The about 1 mM , or about 10 mM , or about 25 mm , or about 50 reactions are from about 1 % to about 99 % enantioselective . mM , or about 100 mM , or about 250 mM , or about 500 mM , The reaction can be , for example , from about 10 % to about or about 1 M . In some embodiments, a reducing agent is 10 90 % diastereoselective , or from about 20 % to about 80 % used in a sub - stoichiometric amount with respect to the diastereoselective , or from about 40 % to about 60 % diaste olefin substrate and the diazo reagent . Cosolvents , in par - reoselective, or from about 1 % to about 25 % diastereose ticular, can be included in the reaction mixtures in amounts lective , or from about 25 % to about 50 % diastereoselective , ranging from about 1 % v / v to about 75 % v /v , or higher. A or from about 50 % to about 75 % diastereoselective . The cosolvent can be included in the reaction mixture , for 15 reaction can be about 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , example , in an amount of about 5 , 10 , 20 , 30 , 40 , or 50 % 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , ( v / v ). 90 % , or about 95 % diastereoselective. The reaction can be Reactions are conducted under conditions sufficient to from about 10 % to about 90 % enantioselective , from about catalyze the formation of a cyclopropanation product. The 20 % to about 80 % enantioselective , or from about 40 % to reactions can be conducted at any suitable temperature. In 20 about 60 % enantioselective , or from about 1 % to about 25 % general, the reactions are conducted at a temperature of from enantioselective , or from about 25 % to about 50 % enanti about 4° C . to about 40° C . The reactions can be conducted , oselective , or from about 50 % to about 75 % enantioselec for example , at about 25° C . or about 37° C . The reactions tive . The reaction can be about 10 % , 15 % , 20 % , 25 % , 30 % , can be conducted at any suitable pH . In general, the reac - 35 % , 40 % . 45 % , 50 % , 55 % . 60 % . 65 % , 70 % , 75 % , 80 % . tions are conducted at a pH of from about 6 to about 10 . The 25 85 % , 90 % , or about 95 % enantioselective. Accordingly reactions can be conducted , for example , at a pH of from some embodiments of the invention provide methods about 6 . 5 to about 9 . The reactions can be conducted for any wherein the reaction is at least 30 % to at least 90 % diaste suitable length of time . In general, the reaction mixtures are reoselective. In some embodiments, the reaction is at least incubated under suitable conditions for anywhere between 30 % to at least 90 % enantioselective . about 1 minute and several hours . The reactions can be 30 conducted , for example , for about 1 minute , or about 5 IV . Examples minutes , or about 10 minutes , or about 30 minutes , or about 1 hour, or about 2 hours, or about 4 hours , or about 8 hours, The present invention will be described in greater detail or about 12 hours , or about 24 hours, or about 48 hours , or by way of specific examples. The following examples are about 72 hours . Reactions can be conducted under aerobic 35 offered for illustrative purposes , and are not intended to limit conditions or anaerobic conditions . Reactions can be con - the invention in any manner . Those of skill in the art will ducted under an inert atmosphere , such as a nitrogen atmo- readily recognize a variety of noncritical parameters which sphere or argon atmosphere . In some embodiments , a sol can be changed or modified to yield essentially the same vent is added to the reaction mixture . In some embodiments , results . the solvent forms a second phase , and the cyclopropanation 40 occurs in the aqueous phase. In some embodiments , the Example 1 . C = C Functionalization by heme enzymes is located in the aqueous layer whereas the Enzyme - Catalyzed Carbenoid Insertion substrates and /or products occur in an organic layer. Other reaction conditions may be employed in the methods of the This example illustrates bacterial cytochrome P450s that invention , depending on the identity of a particular heme 45 are engineered to catalyze highly stereoselective carbene enzyme, olefinic substrate , or diazo reagent. transfers to aryl- substituted olefins, a reaction without a Reactions can be conducted in vivo with intact cells known biological counterpart . expressing a heme enzyme of the invention . The in vivo Creating enzymes that catalyze novel reactions , one of the reactions can be conducted with any of the host cells used hallmarks of evolution , is a huge challenge and nearly for expression of the heme enzymes , as described herein . A 50 unexplored frontier in protein engineering . Carbene trans suspension of cells can be formed in a suitable medium fers to C = C bonds are powerful catalytic methods that lack supplemented with nutrients (such as mineral micronutri- biological counterparts . Stereo - control over these transfor ents , glucose and other fuel sources , and the like ) . Cyclo - mations currently relies on expensive transition metal cata propanation yields from reactions in vivo can be controlled , lysts that require toxic organic solvents and are difficult to in part, by controlling the cell density in the reaction 55 systematically modify or optimize . This example illustrates mixtures . Cellular suspensions exhibiting optical densities variants of cytochrome P450 BM that catalyze an important ranging from about 0 . 1 to about 50 at 600 nm can be used reaction not previously known for this monooxygenase : the for cyclopropanation reactions . Other densities can be use - cyclopropanation of styrene from diazoester reagents with ful , depending on the cell type , specific heme enzymes , or exquisite enantio - and diastereocontrol. As such , this other factors. 60 example demonstrates that existing enzymes can be adapted The methods of the invention can be assessed in terms of for catalysis of synthetically - important reactions not previ the diastereoselectivity and/ or enantioselectivity of cyclo - ously observed in Nature . propanation reaction — that is , the extent to which the reac - Introduction tion produces a particular isomer, whether a diastereomer or The many strategies for functionalizing C = C bonds that enantiomer . A perfectly selective reaction produces a single 65 have evolved in the biological world have captivated the isomer, such that the isomer constitutes 100 % of the product imaginations of chemists who attempt to develop ‘biomi As another non - limiting example , a reaction producing a metic ' catalysts ( J . T . Groves , Proc . Natl. Acad . Sci. U . S . A . US 10 , 208, 322 B2 79 80 100 , 3569 (2003 ) ; R . Breslow , J Biol. Chem . 284 , 1337 chemo -, regio - and stereo - selectivities with which ( 2009 ) ). The reverse of this , developing new biocatalysts cytochrome P450s can insert oxygen atoms into C = C inspired by synthetic chemistry , has received little attention , bonds, whether these enzymes could be engineered to mimic mainly because we poorly understand how to encode a this chemistry was investigated for isoelectronic carbene sequence . Nature' s entire 5 transfer reactions via high - valent iron -enoid species (FIG . desired function in a protein sequence . Nature 's entire 1 ). This example shows that variants of the cytochrome catalyst repertoire has been built with and utilizes physi P450 from Bacillus megaterium (CYP102A1 , or P450BMB ) ologically accessible reagents . Not subject to the same are efficient catalysts for the asymmetric metallocarbene limitations , synthetic chemists have developed powerful mediated cyclopropanation of styrenes . methods for direct C = C functionalization based on transi- 10 Since iron porphyrins are known to catalyze carbene tion metal- catalyzed carbenoid insertions, reactions that are based cyclopropanations ( J. R . Wolf et al. , J . Am . Chem . Soc . used extensively in the synthesis of natural product inter - 117 . 9194 ( 1995 ) ) , whether some common heme proteins mediates and artificial drugs ( H . M . L . Davies et al. , Nature display measurable levels of cyclopropanase ' activity was 1 (2008 ) ). Utilizing high -energy precursors typically first probed . The reaction between styrene and ethyl diazo in the form of acceptor- substituted diazo reagents , these 15 acetate (EDA , FIG . 2 ) , a well- recognized model system for synthetic systems, upon dinitrogen extrusion , form metallo validating new cyclopropanation catalysts , was investigated . carbenoid intermediates that insert into C = C bonds to form Initial experiments showed that optimal formation of the new carbon - carbon centers. Synthetic catalysts , however, desired cyclopropanation products occurred in water in the require expensive transition metals and elaborate ligand 20 presence of a reducing agent (e .g ., sodium dithionite ) under designs for stereocontrol; they also often require toxic anaerobic conditions ( Tables 7 - 10 ). Horseradish peroxidase organic solvents . This example demonstrates combining the (HRP ), cytochrome c ( cyt c ), myoglobin (Mb ) and P450 BM3 high levels of selectivity and ' green ' process conditions all displayed multiple turnovers towards the cyclopropane afforded by enzymes with< th the synthetic power of carbenerbene , products , with HRP, cyt c and Mb showing negligible transfer strategies enabled by transition metal catalysis . 25 enantio - induction and forming the trans cyclopropane with Results Members of the diverse cytochrome P450 enzyme family over 90 % selectivity , which is comparable to the diastereo catalyze myriad oxidative transformations , including selectivity induced by free hemin ( Table 7 ) . Interestingly , hydroxylation , epoxidation , oxidative ring coupling , hetera P450BM3, despite forming the cyclopropane products in low tom release , and heteroatom oxygenation ( E . M . Isin et al . 30 yield , catalyzed the reaction with different diasteroselectiv Biochim . Biophys. Acta 1770 , 314 (2007 ) ) . The majority of ity (cis :trans 37: 63 ) and slight enantio - induction ( Table 11) , transformations encompassed by this broad catalytic scope indicating that carbene transfer and selectivity were dictated are manifestations of the same high -valent iron -oxene inter - by the active site -bound heme cofactor rather than by hemin mediate ( compound I, FIG . 1 ) . Inspired by the impressive released from the protein . TABLE 7 Heme catalysts under anaerobic conditions with sodium dithionite (Na2S204 ) .

0. 1 M KPIpH 8. 0 SARS SAS OEt 5 % MeOH Ph COOEt Ph " COOEt Z= 10 mM Na2S204 RAS + catalyst TRAR Z= Anaerobic PHILI " COOEt Ph . COOEt 30 mM 10 mM cis transtrans

Cat. loading Catalyst Axial ligand (% mol eq) TTN cis: transa % ee cis . % ee transe

Catalase O - Tyr 0 . 16 CPO S - Cys 0 . 40 HRP N - His 1 . 00 7 : 93 - 7 cyt c N - His, S -Met 1 . 00 6 : 94 Mb N -His 1 . 00 6 : 94 P450BM3 5 - Cys 0 . 20 37 :63 Too?7 Hemin 0. 20 6 : 94

" Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b (R , S ) – (SR ). * (R , R ) - ( 8, 8 ). dBioconversion conducted at 0 . 1 M citrate buffer pH = 4 . 0 . US 10 , 208 , 322 B2 81 TABLE 8 Heme catalysts under anaerobic conditions without Na S , 04: SAS OEt 0 . 1 M KPI pH 8 . 0 Pho C OOEt Ph ICOOEt 5 % MeOH + ON + catalyst - ZE Anaerobic R1stRR 30 mM Phil COOEt PAW COOEt 10 mM cis transtrans Cat. loading Catalyst Axial ligand ( % mol eq ) TTN cis : trans % ee cis ee transe Catalase O - Tyr 0 . 16 CPod S - Cys 0 .40 HRP N - His 1. 00 |o cyt c N - His , S -Met 1 . 00 12 8 :92 Mb N -His 1 . 00 0 . 8 11 : 89 wIaIII P450BM3 5 - Cys 0 .20 Hemin 0 .20 11 :89 - 1 " Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b( R , S) - (S ,R ). ( R , R ) – ( S , S ). Bioconversion conducted at 0 . 1 M citrate buffer pH = 4 . 0 .

TABLE 9 Heme catalysts under aerobic conditions with Na S204. 0 . 1 M KPi pH 8 .0 5 % MeOH PASAR sis OEt 10 mM Na2S204 Ph COOEEP COOEt + + catalyst N Aerobic Z= R ^ s . RAR 30 mM Phil. " COOEt Phil COOEt 10 mM cis trans Cat . loading Catalyst Axial ligand (% mol eq ) TTN cis : transa % ee cis % ee trans Catalase O - Tyr 0 . 16 CPOd S -Cys 0 . 40 HRP N - His 1 . 00 12 :88 ??? cytc N -His , S -Met 1 . 00 9 : 91 Mb N -His 1 . 00 7 :93 -13 ??? P450BM3 5 - Cys 0 . 20 13 : 87 Hemin 0 . 20 8 :92 -?5 "Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b ( R , S ) - ( S , R ) . " ( R , R ) - ( S , S ). Bioconversion conducted at 0 . 1 M citrate buffer pH = 4 . 0 .

TABLE 10 Heme catalysts under aerobic conditions without Na S204.

SAS OEt 0 . 1 M KPI pH 8. 0 Ph " COOEt =Z 5 % MOH Ph COOEt + catalyst = RAS Z Anaerobic 30 mM Phllei ICOOEt 10 mM PhD COOEt cis trans Cat. loading Catalyst Axial ligand ( % mol eq ) TTN cis : trans % ee trans Catalase O - Tyr 0 . 16 CPod S -Cys 0 . 40 HRP N - His 1 . 00 cyt c N - His , S -Met 1 .00 US 10 , 208 , 322 B2 83 84 TABLE 10 -continued Mb N - His 1 .00 P450 BM3 5 - Cys 0 . 20 0 . 4 46 :54 - 46 Hemin 0 . 20 "Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b ( R , S ) - ( S , R ) . ( R , R ) - ( S , S ) . dBioconversion conducted at 0 . 1 M citrate buffer pH = 4 . 0 .

10 TABLE 11 TABLE 12 -continued Stereoselective P450BM3 cyclopropanases. Reactions were run in Raw data from P450 BM3 compilation plate screen . Diastereo -and phosphate buffer (pH 8 . 0 ) at room temperature under argon with enantioselectivity were determined by gas chromatography using a chiral 30 mM styrene , 10 mM EDA , 0 . 2 mole % catalyst (with respect to ( B - CDX column as the stationary phase . EDA ) , and 10 mM sodium dithionite . Yields , diastereomeric ratios and 15 enantiomeric excess were determined by GC analysis . P450BM3 Mutations compared to wild - Absolute ?? Catalyst % yielda TTN cis :trans % eecis % eerranse variants type P450BM3 (SEQ ID NO : 1 ) activitya deb ( cis ) WT F87A F87A Hemin 6 : 94 - 1 0 .001704 - 28 57 P450BM3 37 :63 - 27 WT 188L T88L 0 .004522 - 78 23 P450BM3- F87A 6 38 :62 26 - 6 20 WT A328V A328V 0 . 000830 - 100 N / A P450 BM3- T268A 323 1 :99 - 15 - 96 V78A , T1751, A184V , F205C , 0 .001334 - 100 N / A H2 - 5 - F10 59 294 16 :84 - 41 - 63 S226R , H2360 , E252G , H2A10 167 60 :40 - 95 R255S , A290V , L353V H2- 4 - D4 41 206 53 :47 - 79 - 33 139 - 35 V78A , H138Y, T1751, V1781, 0 .001386 - 86 0 C3C 199 71 : 29 - 94 A184V , F205C , S226R , C3C - 1263A 38 190 19 :81 -62 52 - 91 25 H236Q , E252G , R255S , C3C - A328G 186 83 : 17 52 A290V , L353V C3C - T438S 293 92 : 8 - 97 - 66 9 - 10A4 R470 , V78A , K941, P1425 , 0 .004292 - 74 - 20 T1751, A184V , F205C , " Based on EDA S226R , H236Q , E2526 , b ( R , S ) -( S , R ). R255S , A290V , L353V ( R , R ) - ( S , S ). 30 9 - 10A L75W 9 - 10A L75W 0 . 005191 Variant 9 - 10A - TS - F87V - T268A is denoted as C3C . 9 - 10A L7511 9 - 10A L751 0 . 002267 - 3 Other sequence identities are described in Table 12 . 9 - 10A A78F 9 - 10A L78F 0 . 002008 9 - 10A A78S 9 - 10A A78S 0 .005098 - 6 Whether the activity and selectivity of heme- catalyzed 9 - 10A A82G 9 - 10A A82G 0 .002245 - 76 - 7 cyclopropanation could be enhanced by engineering the 9 - 10A A82F1 9 - 10A A82F # VALUE ! N / A N / A protein sequence was determined . P450 BM3 is a well - stud - » 9 - 10A A820 9 - 10A A820 0 . 002487 - 74 16 ied , soluble , self- sufficient (heme and diflavin reductase 9 - 10A A821 9 - 10A A821 0 .001031 - 100 N / A 9 - 10A A825 9 - 10A A82S 0 .001483 - 8214 domains are fused in a single polypeptide ), long -chain fatty 9 - 10A A82L4 9 - 10A A82L 0 .000591 - 100 N / A acid monooxygenase . More than a decade of protein engi 9 - 10A F87A 9 - 10A F87A 0 .001701 - 61 - 10 neering attests to the functional plasticity of this biocatalyst 1 9 - 10A F87vl 9 - 10A F87V 0 . 000000 N / A N / A ( C . J . C . Whitehouse et al. , Chem . Soc. Rev. 41, 1218 40 9 -10A F8714 9- 10A F871 0 .000983 - 100 N / A ( 2012 ) ) . Thousands of variants that exhibit monooxygenase 9 - 10A F87L 9 - 10A F87L 0 .000710 - 100 N / A 9 - 10A T88019 - 10A 788C 0 . 002516 3 activity on a wide range of substrates have been accumu 9 - 10A T2608 9 - 10A T260S 0 .004259 - 82 lated from using directed evolution to engineer cytochrome 9 - 10A T260N 9 - 10A T260N 0 . 003882 - 77 P450BM3 for synthetic applications (I . C . Lewis et al ., 45 9 - 10A T260L ! 9 - 10A T260L 0 .006173 - 77 Chimia 63 , 309 ( 2009 ) ). Some of these variants were tested 9 - 10A A328V 9 - 10A A328V 0 .006471 - 68 by chiral gas chromatography for altered cyclopropanation 9 - 10A A328M 9 - 10A A328M 0 . 005180 diastero - and enantioselectivity . A panel of 92 P450BM3 9 - 10A A328F1 9 - 10A A328F 0 . 002009 49 - 1A R470 , V78T , A82G , F87V , 0 .001874 - 75 variants , chosen for diversity of activity and protein K941, P142S , T1751 , A184V , sequence , was screened in E . coli lysate for the reaction of F205C , S226R , H236Q , styrene and EDA under aerobic conditions in the presence of 30 E252 , R255S , A290V , sodium dithionite ( Tables 12 and 13 ) . The ten most prom A328L , L353V ising hits were selected for purification and subsequent 35 - 7F R470 , V78F, A82S , K941 , 0 .004514 - 73 -52 characterization under standardized anaerobic reaction con P142S , T1751, A184V, ditions ( Tables 11 and 14 ) . F205C , S226R , H236Q , 55 E252G , R2555 , A290V , A328L , L353V TABLE 12 53 -5H 9 - 10A A78F, A82S , A328F 0 . 002840 - 80 2 7 - 11D 9 - 10A A82F , A328V 0 .036840 - 24 - 28 Raw data from P450BM3 compilation plate screen . Diastereo - and 49 - 9B enantioselectivity were determined by gas chromatography using a chiral R47C , V78A , A82G , F87V , 0 .000000 N / A N / A ( B - CDX column as the stationary phase . K941, P142S , T1751, A184V , - 60 F205C , S226R , H236Q , E252G , R255S , A290V , Mutations compared to wild - Absolute ee A328L , L353V variants type P450BM3 (SEQ ID NO : 1 ) activitya deb ( cis ) 41 - 5B R470 , V78F , A82G , K941, 0 .008391 - 77 - 17 CYP102A3 N / A 0 .004053 - 74 - 8 P142S , T1751, A184V, CYP102A2 N / A 0 .002963 - 76 - 36 F205C , S226R , H236Q , CYP102A1 None 0 . 002240 - 81 7 65 E252G , R2555 , (P450BM3 ) A290V , A328V, L353V US 10 ,208 , 322 B2 85 86 TABLE 12 - continued TABLE 12 - continued Raw data from P450BM3 compilation plate screen . Diastereo - and Raw data from P450BM3 compilation plate screen . Diastereo - and enantioselectivity were determined by gas chromatography using a chiral enantioselectivity were determined by gas chromatography using a chiral (B - CDX column as the stationary phase . ( B -CDX column as the stationary phase . P450 BM3 Mutations compared to wild - Absolute ?? P450BM3 Mutations compared to wild - Absolute ?? variants type P450BM3 (SEQ ID NO : 1 ) activitya deb ( cis ) variants type P450 BM3 ( SEQ ID NO : 1 ) activitya deb (cis ) 13 -701 9 - 10A A78T , A328L 0 .005493 - 73 - 43 9073 12 - 10C R47C , V78A , A82G , F87V , 0 . 004566 | - 21 9 - 10A C47R , A78L , F875 , 0 . 007258 - 79 - 5 K941, P142S , T1751, A184V , 10 194K , A180V , V184T , F205C , S226R , H236Q , G315S , A330V, Y345C E252G , R255S , A290V , B13B13 9 - 10A C47R , A78L , F87A , 0 . 002246 - 61 - 14 A328V , L353V 194K , V184T, 1263M , 77 -9H 9 - 10A A78T, A82G , A328 L 0 . 003053 - 73 - 34 G315S , A330V 11 -8E R470 , V78A , F87V , K941, 0 .001453 - 77 15 BISYN3 9 - 10A C47S , N70Y, A78L , 0 .002705 - 76 - 23 P142S , T1751, A184V , 15 F87A , 1174N , 194K , V184T , F205C , S226R , H236Q , 1263M , G315S , A330V E252G , R255S , H2 - 4 - D4 9 - 10A TS F87V , L75A , 0 . 052439 A290V, A328L , L353V M177A , L181A , T268A , 1 - 1264 9 - 10A A82L , A328V 0 .003884 - 70 - 19 L437A 29 - 3E R470 , V78A , A82F, K941, 0 .003425 E12 A8773 9 - 10A C47R , A78L , F87V , P142S , T1751, A184V , 0 .001990 - 65 - 52 F205C , S226R , H236Q , 20 194K , A111V , V1411 , E252G , R255S , A180V , V184T, A290V, A328F , L353V G315S , A330V 29 - 10E R470 , V78F , A826 , K941, 0 . 001935 - 70 16 GICA4 T180A 9 - 10A C47R , F81W , A82S , 0 .004925 - 78 12 P142S , T1751, A184V , F87A , 194K F2050 , S226R , H236Q , H2- 8 - C72 9 - 10A TS F87V , L75A , 0 .000808 - 100 N / A E2526 , R255S , 25 L181A A290V , A328F, L353V CH - F8 9- 10A L51A , C47A , F87V , 0 . 001126 - 100 N / A 68- 8F1 9 - 10A A78F , A82G , A328L 0 . 004127 - 72 - 32 35E116 R470 , V78F , A82S , K941, 0 .003600 - 71 - 14 194K , L181A , C205F , P142S , T1751, A184V , S254R , 1366V , F205C , S226R , L437A , E442K H236Q , E2526 , R255S , 30 H2- 4 - H52 9 - 10A TS F87V , L75A , 0 . 001229 A290V , A328F , L353V , M177A , L181A E4646 , 1710T SA9 9 - 10A C47R , F81W , A821, 0 .004170 - 81 11 19A126 35E11 L521 , L188P, 1366V 0 .006909 - 70 - 27 F87A , 194K , A180T, A197V ETS86 35E11 L521, 1366V 0 .003966 - 79 - 19 ManA10 9 - 10A C47R , F81S , A82V , 0 .006340 ( 11 -3 )6 35E11 L521, A74S , L188P , 0 .005633 - 76 - 39 1366V F87A , 194K , A180T, A197V ( 7 - 7 ) 35E11 L521, A74E , S82G , - 77 -9 35 Manl 9 - 10A C47R , F81L , A82T , 0 . 003053 A184V , L188P , 1366V 0. 010499 F87A , 194K H2A10 9 - 10A TS F87V , L75A , 0 . 066422 -8 -94 MB2 9 - 10A C47R , F81W , A821, 0 . 003282 L181A , T268A F87A , 194K SL2 -6F8 R47C , L521 , V78F , A82S , 0 . 000778 - 100 N / A HA62 9 - 10A C47R , F81A , A82L , 0 .003375 - 81 -5 K941, P142S , T1751 , A184V , F87A , 194K F205C , S226R , H236Q , 9 - 10A TS V78A , P142S , T1751, E252G , R255S , A290V , 0 .001920 A328L , K349N , L353V , A184V , S226R , H236Q , 1366V , E464G , 1710T E252G , A290V , A12SL - 17 - 4 R47C , L521, A74E , V78F , 0 .010935 - 80 6 L353V , 1366V, E442K A82S , K941, P142S , T1751, 9 - 10A TS 9 - 10A TS F87A 0 . 001546 A184V , L188P , F205C , 45 F87A S226R , H2360 , E2526 , 25F7 9 - 10A C47R , A74F, A78S , 0 .001829 - 81 43 R255S , A290V , A328F , F87A , 1282K , C205F , S255R L353V , 1366V , E464G , 2404 9 - 10A C47R , A741, A78L , 0 .000783 - 100 N / A 1710T H2 - 2 - A12 F87A , 194K , C205F, S255R 9 - 10A TS F87V , L75A , 0 .003042 - 75 - 11 5A1 0 .002471 L181A , L437A 9 - 10A M30T , C47R , A74F, Al2RM - 2 - 8 R47C , L521, A74E , V78F , 0 . 007705 - 77 - 13 A78S , 194K , C205F , S255R , A82S , K941, P142S , T1751, Q310L , 1366V, E442K A184S , L188P , F205C , 8B3 9 - 10A M30T, C47R , A74F, 0 .001315 - 100 N / A S226R , H236Q , A78S , 194K , C205F , C255R , E252G . R255S . L310Q , Q323L , 1366V , A290V , A328F, L353V , N381K , R398H , E441K 1366V, E464G , 1710T H2 - 5 - F10 9 - 10A TS F87V , L75A , 0 .141237 - 46 - 56 Determined by GC analysis on a chiral B -CDX column . 1263A , T268A , L437A " Reported as the sum of the area of the cyclopropane peaks over the area of the internal 13C9R1 L521, 158V , L75R , F87A , 0 .001980 - 100 N / A standard . H100R , S106R , F107L , " Diastereomeric excess = ( [ cid ] – [trans ] )/ ( [cis ] + [trans ] ). A135S . A184V . N239H . 60 ( R , S ) - ( S , R ) . S274T, L3241, V340M , ' P. Meinhold et al. , Adv. Synth . Catal . 348, 763 ( 2006 ) . 21. C . Lewis et al ., Chembiochem : a European journal of chemical biology 11, 2502 1366V , K434E , (2010 ) . E442K , V4461 J . C . Lewis et al. , Proceedings of the National Academy of Sciences of the United States 22A3 13C9R1 F1621 E434K 0 .004053 - 70 4 of America 106 , 16550 (2009 ) . K442E 1446V 4M . W . Peters et al. , J . Am . Chem . Soc. 125 , 13442 ( 2003 ) . 2063 9 - 10A A78L , F87A , V184T , 0 .004257 - 78 - 15 65 A . Glieder et al. , Nat. Biotechnol. 20 , 1135 ( 2002 ) . G315S , A330V R . Fasan et al ., Angew . Chem ., Int Ed . 46 , 8414 (2007 ) . US 10 , 208 , 322 B2 87 88 TABLE 13 Five of the ten selected P450s showed improvements in activity ( > 100 TTN ), a comprehensive range of diastereo Raw GC screening data for the selectivities with cis : trans ratios varying from 9 :91 to 60 : 40 , chimeric P450s in the compilation plate . and impressive enantioselectivities (up to 95 % ee, Table 14 ). Chimeric P450s (heme Absolute ee 5 For example, variant H2 - 5 -F10 , which contains 16 amino P450 domain block sequence ) activity des (cis ) acid substitutions from wild type , catalyzes 294 TTN , CYP102A1 11111111 0 .001704 - 28 56 equivalent to - 58 % yield (with respect to EDA ) under these (P450BM3 ) F87A " conditions. This represents a 50 - fold improvement in TTN CYP102A2 22222222 N / A N / A N / A F88A over wild type P450BM3. Furthermore , mutations affect both CYP102A3 33333333 N / A N / A N / A the diastereo - and enantioselectivity of cyclopropanation : F88A H2 - 5 -F10 favors the trans cyclopropanation product ( cis : 5R12 32312231 0 . 008625 58 19 9R12 12112333 0 .0042707 58 24 trans 16 :84 ) with 63 % eerone, while variant H2A10 , which 12R12 12112333 0 . 0701514 32 - 49 catalyzes up to 167 TTN , demonstrates reversed diastereo C1D11R12 21113312 0 . 007138 selectivity ( cis: trans 60 : 40 ) with high enantioselectivity C2B12R12 32313233 0 . 005914 (95 % ee cis ). C2C12R12 21313111 0 . 006226 C2E6R12 11113311 0 . 008731 25 The variant H2A10 was used to verify the role of the C2G9R12 22213132 0 . 007975 15 enzyme in catalysis and identify optimal conditions ( Table C3D10R12 22132231 0 . 004898 - 16 15 , FIGS . 3 and 4 ) . Heat inactivation produced diastereo C3E4R12 21313311 0 .007893 17 and enantioselectivities similar to those obtained using free F3H12R12 21333233 0 . 005586 - 56 - 17 20 hemin , consistent with protein denaturation and release of F6D8R12 22313233 0 . 008088 - 76 -6 the cofactor. Complete inhibition was achieved by pre C3B5R12 23132233 0 . 014722 - 814 incubating the bioconversion with carbon monoxide , which X7R12 22312333 0 .017305 - 4 - 34 irreversibly binds the reduced P450 heme, confirming that " Reported as the sum of the area of the cyclopropane peaks over the area of the internal catalysis occurs at the active site . Air inhibited the cyclo standard . propanation reaction by about 50 % , showing that dioxygen Diastereomeric excess = ([ cis ] – [ trans] )/ ([ cis] + [trans ]) . ( R , S ) - ( S , R ) . and EDA compete for reduced Fe " . Cyclopropanation was ' C . R . Otey et al. , PLOS Biol. 4 , 789 ( 2006 ) . also achieved with NADPH as the reductant, confirming that M . Landwehr et al ., Chem . Biol. 14 , 269 (2007 ) . the novel activity can also be driven by the endogenous Site - directed recombination of three bacterial cytochrome P450s was performed with electron transport machinery of the diflavin - containing proteinsequence structure crossover . Seven sites crossoverchosen to sitesminimize where the chosen number resulting of dismpted in eight contacts sequence within blocks the. 3 o reductase domain . The presence of a reducing agent in The numbering refers to the identity of the parent sequence at each block . For example , sub -stoichiometric amounts proved essential for cyclopro " 12312312 ” refers to a sequence containing block 1 from P450 1 , block 2 from P450 2 , block 3 from P450 3 , etc. panation ( Table 16 ) , implying that the active species is Fell rather than the resting state Fe . TABLE 14 Stereoselective P450BM3 based cyclopropanases.

0. 1 M KPI pH 8. 0 s / R 5 % MeOH SAS OEt 10 mM Na2S204 Ph" COOEt Ph " COOEt + ON=ZEZ P450 ^ Anaerobic st RAR 30 mM 20 uM " COOEt Philli COOEt 10 mM 0 .2 % mol eq cis trans (wrt EDA ) trans P450 % yield " TTN cis: trans % ee cis % ee transa

WT 37: 63 WTF87A 1 . 2 37 :63 H2A10 33 . 4 167 60 : 40 H2 - 4 - 14 41. 2 206 53 : 47 H2 - 5 - F10 58 . 8 294 16 : 84 C2C12R1 1 . 6 36 :64 C3E4R1 1 . 6 43 :57 X7R1 2 . 4 33 :67 12R1 17 : 83 C2E6 R1 23 27 : 73 C2G9 R1 48 240 9 :91 7 - 11D 160 35 :65 abased on EDA " Diastereomeric ratios and enantiomeric excess were determined by GC analysis . ( R , S ) - ( S , R ). d (R . R ) - ( S, S ). US 10 , 208 , 322 B2 89 TABLE 15 Controls for P450 based cyclopropanation using variant H2A10 .

0 . 1 M KPI pH 8 .0 OEt 5 % MeOH SAR A 10 mM5% MeOHNa2S204 Ph COOEt Ph PICOOEt + + P450 ZEZ H2A10holo . RAR 10 mM ON Pb1111 20 mM 20 UM COOEt Phil COOEt 0 . 2 % mol eq cis trans (wit styrene )

Conditions TTN % inhibition cis : transa % ee cis ee trans© Complete System ( CS ) 70 :30 - 78 CS -Na2S204 + NADPH - 55 61 : 39 CS -Na2S204 + NADH - 62 53 : 47 CS- Na2S204 - 100 *|Chi CS- P450 - 100 CS + CO - 100 Boiled P450 + 45 16 : 84 H2A10 heme

TABLE 16 Highly active variants H2A10 , H2- 5- F10 and H2 -4 -D4 35 have three to five active site alanine substitutions with Effect of concentration of sodium dithionite on cyclopropane yield . respect to 9 - 10A - TS - F87V (12 mutations from wild type ), which itself shows negligible cyclopropanase activity . These variants demonstrate significant differences in TTN , diaste OEt 40 reoselectivity , and enantioselectivity ( Table 11 ) . To better ON understand how protein sequence controls P450 -mediated cyclopropanation , 12 new variants were constructed to 20 mM ON assess the contributions of individual alanine mutations to 10 mM catalysis and stability ( Table 17 ) . T268A is key for achieving 45 high levels of cyclopropanation activity , and this mutation 0 . 1 M KPi pH 8 . 0 P450 5 % MeOH alone converts inactive 9 - 10A - TS - F87V into an active Na2S204 + H2A10holo Anaerobic cyclopropanase . Variant 9 - 10A - TS - F87V - T268A (denoted Variable C3C ) is a competent cyclopropanase ( 199 TTN ) , displays 20 uM 0 . 2 % mol eq 50 strong preference for the cis product (cis :trans 71: 29 ) , forms (wrt EDA ) both diastereomers with over 90 % ee , and is as stable as wild - type P450R12 . Other active site alanine mutations tune COOEt the product distribution . Notably , the addition of 1263A to C3C reverses diastereoselectivity ( cis :trans 19: 81 ). The effects of similar mutations introduced in the poorly active Phar wild type P450BM3 were also investigated ( Table 18 ). Impressively , P450BM3 - T268A ,with a single mutation , is an [Na2S204 ]/ mM [cyclopropanes ]/mM TTN active cyclopropanase ( 323 TTN , Table 11 ) with exquisite 0 60 trans - selectivity (cis :trans 1 : 99 ) and high enantioselectivity 2 . 59 129 2 . 72 136 for the major diastereomer ( - 96 % eetrans , FIG . 2 ). Whereas 10 3 . 34 167 C3C is a cis - selective cyclopropanase , identical active site 20 3 . 13 156 mutations in wild type P450BM3 result in a trans - selective 50 2 .79 140 enzyme ( Table 18 ), demonstrating that mutations outside of 100 2 . 71 136 the active site can also influence the stereochemical out come. US 10 ,208 , 322 B2 91 TABLE 17 Mutational analysis of alanine substitutions on 9 - 10A TS F87V .

0 . 1 M KPi pH 8 . 0 OEt 5 % MeOH Phone COOEt Ph COOETC = 10 mM Na2S204 A . COM + ON + 9 - 10A TS F87y variant R1st RAR ON 30 mM PH COOEt Phì24COOP coi?t 10 mM 20 uM cis trans 0 . 2 % mol eq (wrt EDA )

Mutations relative to 9 - 10A cis : % ee % ee P450 (Holo ) L75A M177A L181A 1263A T268A 1437A T TN transa cish transe T50 (° C .) 9 - 10A TS F87V No No No No No No 35 :65 - 41 - 8 59 . 5 9 - 10A TS F87V Yes No No No No No 42 : 58 - 59 - 11 52 . 3 L75A 9 - 10ATS F87V No No Yes No No 5 41: 59 - 27 - 7 53 . 3 L181A No No 9 - 10A TS F87V No No YesYes NoNo No 8 29 :61 - 31 - 39 39 5555 .. 4 1263A 9 - 10A TS F87V No No No No Yes No 199 71: 29 - 94 94 -- 91 55 . 2 T268A (C3C ) 199 C3C 1263A No No No Yes Yes No 190 19 : 81 54 . 0 C3C L181A No No Yes No Yes No 159 56 : 44 -92 50 . 8 H2A10 Yes No Yes No Yes No 167 60 : 40 48 . 9 C3C L181A No No Yes Yes Yes No 203 14 : 86 50 . 9 1263A C3C L181A No No. Yes No Yes Yes 180 27: 73 - 74 8 48 .4 L437A - 74 - 98 C3C L181A No No Yes Yes Yes Yes 1263A L437A 218 9: 91 -55 - 96 48 .2 4H5 Yes Yes Yes No No No 32 :68 -9 0 49 . 4 C3C 1263A No No No Yes Yes Yes 267 16 : 84 - 59 - 89 50 . 4 L437A H2 - 5 - F10 Yes No No Yes Yes Yes 294 16 :84 - 41 - 63 47 . 5 H2 - 4 - D4 YesYes YesNo Yes Yes 20653 : 47 - 79 - 3346 .4 Diastereomeric ratiosand enantiomeric excess were determined by GC analysis . b ( R , S ) - ( S , R ) . ( R , R ) - ( S , S ).

TABLE 18 Introducing variant 9 -10A - TS -F87V - T268A related active site mutations in wild type P4508M3. 0 . 1 M KPI pH 8 .0 SAS OET 5 % MeOH 10 mM Na2S204 COOEt Ph " COOEt + ON + Wild - Type ON variant 30 mm 10 mM Phº coOEt PH COOFt 20 AM cis trans 0 . 2 % mol eq (wrt EDA ) Mutations relative to wild -type P450BM3 P450 (Holo ) V78 F87 T268 1263 TTN cis: transa % ee cist % ee transº T50 (° C .)

WT 37 :63 - 10 O 56 . 0 WT- F87A 38 :62 O 53. 0 WT- F87V | 9 30 :70 52 . 9 I| 9 WT- T268A 323 1 : 99 O 53. 6 WT- F87V / T268A - 274 32 :68 - 77 - 99 52 . 0 WT- V78A/ F87V / T268A - 190 32 : 68 - 70 - 20 50 . 8 A WT- F87V / 1263A / T268A |- VAA2467938: -9450 . 0 "Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b (R , S) - (S ,R ). ( R , R ) - ( S , S ) . US 10 , 208 , 322 B2 93 94 Since the design of cis - selective small- molecule catalysts L437 , T438 ) were chosen for individual site - saturation for diazocarbonyl- mediated cyclopropanations has proven mutagenesis (see , Materials and Methods ). Substitutions more challenging than their trans counterparts ( A . Caballero A328G , T438A , T438S and T438P all afforded enhanced et al. , European Journal of Inorganic Chemistry , 1137 cis -selectivity ( Table 19 ) . Notably A328G also reversed the ( 2009 ) ) , whether further active site engineering of P450 m2 enantioselectivity for the cis - diastereomer ( Table 11 ) . C3C could provide robust cis -selective water - compatible cata - T438S displayed the highest diastereo - and enantioselectivi lysts to complement existing organometallic systems was ties ( cis : trans 92 : 8 and - 97 % eecis ) and maintained activity investigated . Five active site residues (L181 , 1263 , A328 , comparable to C3C ( Table 11 ) . TABLE 19 Cyclopropanation activity of selected C3Cheme active site variants .

0 .1 M KPI pH 8. 0 5 % MeOH s ^ s OEt C3Cheme active site mutant 10 mM Na2S204 PA C OOEt Ph ICOOF + + ON 7 active R ON 30 mM 20uM PhD 10 mM 0 . 2 % mol eq COOEt Ph !! ! COOET (wrt EDA ) cis trans

P450heme Yield ( % ) TTN cis: trans % ee cise % ee transd 9 - 10A TS 286 71: 29 - 92 - 88 F87V T268A ( C3C ) C3C - L1816 234 59 :41 - 89 C3C - A328G 186 83 : 17 -45 C3C - L437F 265 53 :47 - 85 C3C - L4370 minm 148 53 : 47 W C3C - L4376 290 54 : 46 - 91 C3C - L437A 194 38 :62 -11 C3 - T438A 54 91 : 9 - 75 C3C - T438G 15 73 :27 -59 C3C - T4385 293 92 : 8 C3C - T438Q 206 38: 62 67 V C3C - T438P 161 90 : 10 - 91 - 50

based on ED " Diastereomeric ratios and enantiomeric excess were determined by GC analysis . (R , S ) - ( S ,R ). R ) - ( S . S ) . Variant 9 - 10A - TS - F87V - T268A is denoted as C3C .

C3C exhibits Michaelis -Menten kinetics (FIG . 5 and Table 20 ) with similar Ky values for the olefin ( ~ 1 . 5 mM ) and the diazocarbene ( ~ 5 mM ) . The relatively high KM 45 values reflect the lack of evolutionary pressure for these enzymes to bind these substrates . C3C exhibits a notable kcat for cyclopropanation of 100 min - 1 , comparable to the kes of many native P450s for hydroxylation , but about fifty times slower than P450BM3 - catalyzed fatty acid hydroxylation ( Table 21) . When used at 0 . 1 mol % equivalent, C3C 50 catalyzed cyclopropanations reached completion after 30 minutes. Adding more EDA equivalents leads to enhanced turnovers for cyclopropanes , with preserved C3C stereose lectivity ( Table 22 ) , confirming catalyst integrity and imply ing that the reaction stops because of EDA depletion rather than due to mechanistic inactivation . TABLE 20 Michaelis -Menten parameters for P450 cyclopropanases variant 9- 10A - TS -F87V T268A (herein called C3C ) . kearl (KM - EDA * Kut- styrene Kead KM -EDA kcal Kal- sgrene KM - styrene) catalyst keat (min - 1) KM(mm -EDA ) (mm ) (81M - ) ( 8 + m “ ) (s + M -1M -1 ) C3Creme 100 24 5. 2 + 3 .5 1. 4 + 0 .5 320 1, 100 2 .1 x 105 US 10 ,208 , 322 B2 95 96 TABLE 21 Kinetic parameters for wild - type cytochrome P450s acting on their native substrates and for an engineered variant of P450BM3 (propane monooxygenase , PMO ) acting on the non -native substrate propane . kcal/ KM -EDA P450 Substrate kcat (min - 1) KM -EDA (MM ) (s - 1 M - ) CYP153A6 Octane 75 0 .32 3 . 900 P450BM3 Lauric 5140 0 . 29 3. 0 x 105 acid 10 PMO3 Propane 450 0 .17 4. 4 x 104 IM . M . Chen et al. , Advanced Synthesis & Catalysis 354, 964 ( 2012 ). 2M . A . Noble et al. , Biochemical Journal 339 ( Pt 2 ), 371 ( 1999 ). R . Fasan et al. , . J. Mol. Biol . 383 , 1069 ( 2008 ) .

TABLE 22 Effect of EDA addition at t = 30 min on variant 9 - 10A - TS -F87V - T268A -catalyzed cyclopropanations. 0 . 1 M KPIpH 8 . 0 A SAS COOEt C3Cheme 5 % MOH 10 mMN Ph COOEt Ph ' COOEt Phº 4 || + 20 AM + 30 mM N2 0 .2 % mol eq RAS Table $ 17 (wrt EDA) Ph ” “ COOEt Phì COOEL cis trans % ee Conditions TTN cis :transa ciss % ee trans 10 mM EDA added at t = 0 273 = 2 .5 72 : 28 - 90 10 mM EDA added at t = 0 425 + 17 73: 27 - 93 - 89 + 10 mM EDA at t = 30 min TTN values are reported as the mean of triplicates standard deviation . " Diastereomeric ratios and enantiomeric excess were determined by GC analysis . b (R , S ) – (S , R ). ( R , R ) - (S , S ) .

To assess substrate scope of P450BM3- catalyzed cyclo TABLE 23 - continued propanation , the activities of seven variants against various olefins and diazo compounds were investigated ( Tables 23 - 27 ) . P450 cyclopropanation is robust to both electron 40 0 . 1 M KPi pH 8 .0 donating ( p - vinylanisole , p - vinyltoluene ) and electron -with 5 % MeOH drawing ( p - trifluoromethylstyrene) substitutions on styrene , 10 mM Na2S204 P450 and 7 - 11D demonstrated consistent cis - selectivity for these Anaerobic substrates. The P450s were also active on 1 , 1 -disubstituted 45 20 M olefins ( e. g. , a -methyl styrene ), with chimeric P450 ( 0 . 2 mol % ) C2G9R1 forming cyclopropanes in 77 % yield (with respect to EDA ) . The P450s were only moderately active with COOR t -butyl diazoacetate as substrate ( 30 TTN ) , forming the trans product with > 87 % selectivity and offering no advan - 50 N tage over free hemin ( Table 27 ) . However, for reactions involving EDA and aryl- substituted olefins , the P450s con sistently outperformed the free cofactor in both activity and stereoselectivity . P450 % yield TTN cis :trans % ee cis % ee transa TABLE 23 7 - 11D 104 54 : 46 0 . 3 N / A Substrate scope of P450 cyclopropanases: p -methylstyrene + EDA . H2 - 5 - F10 222 11: 89 14 . 9 N / A C2G9 R1 92 10 . 90 8 . 9 N / A H2A10 50 43 : 57 - 84 . 3 N / A 60 9 - 10A TS F87V 228 78 : 22 - 81. 4 N / A UET T268A (C3C ) Hemin 7 37 6 :94 94 -- 1 . 6 NA GC ( cyclosil - B column 30 mx 0 . 32 mm , 0 . 25 um film ): oven temperature = 100° C . for ON 5 min , 5º C ./ mm to 200° C ., 20° C ./ mm to 250° C ., 250° C . for 5 mm . Elution times: 30 mM ZZ o cis -cyclopropanes (21 . 03 and 21 . 18 min ), trans- cyclopropanes (22 . 71 min ) . 10 mM atrans- enantiomers did not resolve . US 10 , 208 , 322 B2 97 98 TABLE 24 U810208:52 B2 TABLE 25 a Substratere scopealso of P450 remontucyclopropanases : p - vinylanisole + EDA . Substrate scope of P450 cyclopropanases p - ( trifluoromethyl) styrene .

OEt OEt + +

Meo ONZEZ 10 F3C ON 30 MM 10 mM 30 mM 10 mM 0 . 1 M KPIpH 8 . 0 0 . 1 M KPI pH 8 . 0 5 % MeOH 5 % MOH 10 mM Na2S204 10 mM Na2S204 P450 P450 Anaerobic Anaerobic 20 UM 20 uM ( 0 . 2 mol % ) ( 0 . 2 % mol eq) COOEt 20 COOET

Me01 25 F C P450 % yield TTN cis :trans % ee cis % ee transa P450 % yield TTNa cis: trans % ee cis % ee trans 7 - 11D 297 70 : 30 - 27 N / A 7 - 11D 120 76 : 24 31 59 H2- 5 - F10 364 11 : 89 38 N / A H2- 5 - F10 40 1 98 26 :74 72 - 65 C2G9 R1 196 10 :90 N / A 30 C269 R1 18 89 10 : 90 H2A10 80 40 :60 - 75 N / A H2A10 47 26 :74 - 24 22 9 - 10A TS F87V 214 48 :52 - 44 N / A 9 - 10A TS F87V 211 39 :61 54 - 93 T268A (C3C ) T268A ( C3C ) Hemin 96 7 : 93 0 N / A Hemin 11 : 89 1 35 " Assumed the same detector response factor as for ethyl 2 - ( 4 -methylphenyl ) cyclopropane GC oven temperature = 110° C . for 8 min , 2° C . /mM to 180° C . then 180° C . for 30 min , 1 - carboxylate . GC ( cyclosil - B column 30 mx 0 .25 mm , 0 . 25 um film ) :oven temperature 175 kPa . Cyclosil- B column ( 30 mx 0 . 25 mm , 0 .25 um film ). Elution times : cis = 110° C . for 8 min , 2° C . /min to 180° C . then 180° C . for 30 min , 175 kPa . Elution cyclopropanes (38 . 74 and 39. 52 min ) , trans -cyclopropanes (43 .07 min ) . times: cis - cyclopropanes ( 27 . 26 and 28 . 11 min ), trans - cyclopropanes ( 30 . 78 and 30 . 99 " Baseline resolution could not be achieved for the trans - enantiomers. min ) . TABLE 26 Substrate scope of P450 cyclopropanases: a -methyl styrene .

0 . 1 M KPIpH 8 . 0 P450 OEt 5 % MeOH Mell COOEt Eto00 \' Me (2 ) 10 mM Na2S204 Ph Ph + ON + 20 uM ( 0 . 2 % mol Anaerobic 30 mM Phim COOEt Etoo Ph 10 mm eq ) Me EtooCADMe (E )

P450 % yield TTN Z : E % ee ( Z ) % ee ( E )" 7 - 11D 157 41 : 49 N / A H2- 5 - F10 329 21 : 79 N / A C2G9 R1 387 16 :84 N / A H2A10 168 19 :81 N / A 9 - 10A TS F87V 127 16 :84 N / A T268A (C3C ) WT F87V T268A 312 7 : 93 N / A Hemin 77 24 : 76 N / A

GC oven temperature = 100° C . for 5 mm , 1° C ./ mm up to 135º C ., 135° C . for 10 min , 10° C ./ mm up to 200° C . , 200° C . for 5 mm . Cyclosil - B column ( 30 m x 0 . 32 mm , 0 . 25 um film ) . Elution times : Z -cyclopropanes ( 34 . 96 and 35 . 33 min ), E - cyclopropanes ( 39 . 34 and 39 .61 min ) . atrans - enantiomers did not separate to baseline resolution . US 10 , 208 , 322 B2 99 100 TABLE 27 recorded on either a Varian Mercury 300 spectrometer (300 MHz and 75 MHz, respectively ) , or a Varian Inova 500 MHz Substrate scope of P450 cyclopropanases : t -butyl diazoacetate . (500 MHz and 125 MHz, respectively ) , and are internally referenced to residual solvent peak . Data for ' H NMR are reported in the conventional form : chemical shift ( 8 ppm ) , multiplicity (s = singlet , d = doublet , t = triplet , q = quartet , m = multiplet, br = broad ) , coupling constant (Hz ) , integration . Data for 13C are reported in termsof chemical shift (d ppm ) and multiplicity . High -resolution mass spectra were 30 mM ?N 10 obtained with a JEOL JMS - 600H High Resolution Mass 10 mM Spectrometer at the California Institute of Technology Mass Spectral Facility . Reactions were monitored using thin layer 0 . 1 M KPI pH 8 . 0 chromatography (Merck 60 silica gel plates ) using an UV 5 % MeOH lamp for visualization . Optical rotation was measured using 10 mM Na2S204 15 a JASCO P - 2000 Polarimeter. P450 Anaerobic Gas chromatography (GC ) analyses were carried out 20 UM using a Shimadzu GC - 17A gas chromatograph , a FID detec ( 0 . 2 mol % ) tor, and J & W scientific cyclosil - B columns ( 30 mx0 .32 mm , 0 . 25 um film and 30 mx0 .25 mm , 0 .25 um film ). High 20 performance liquid chromatography (HPLC ) was carried out using an Agilent 1200 series , an UV detector, and an Agilent XDB -C18 column ( 4 .6x150 mm , 5 m ) . Cyclopropane prod uct standards for the reaction of ethyl diazoacetate (EDA ) with styrene ( ethyl 2 -phenylcyclopropane - 1 -carboxylate ) 25 and a -methylstyrene ( ethyl 2 -methyl - 2 - pehnylcyclopro P450 % yielda TTN cis : trans pane - 1 -carboxylate ) were prepared as reported (A . Penoni et WT F87V T268A 1 . 4 4 : 96 al. , European Journal of Inorganic Chemistry, 1452 (2003 ) ) . 7 - 11D 11 54 13 :87 These standards were used in co - injection experiments to H2- 5 - F10 18 90 3 : 97 determine the authenticity of enzyme -catalyzed cyclopro H2A10 24 120 3 :97 30 panes. Authentic P450 catalyzed cyclopropane samples were 9 - 10A TS F87V 0 . 3 3 : 97 T268A (C3C ) also prepared as described below and were characterized by Hemin 20 100 4 :96 NMR (+ H and 'SC ) and mass spectrometry . Azides 5 and 8 , and benzosultam standards 6 and 9 were prepared as " Assumed the same detector response factor as for ethyl 2 - ( 4 -methylphenyl ) cyclopropane 1 - carboxylate . GC ( cyclosil - B column 30 mx 0 . 32 mm , 0 . 25 um film ) : oven temperature reported ( J . V . Ruppel et al. , Org . Lett . 9 , 4889 (2007 ) ) . = 100° C . for 5 min , 5° C /mM to 200° C . , 20° C . /mM to 250° C . , 250° C . for 5 min . Elution tan times : cis - cyclopropanes ( 21 .66 min ) , trans - cyclopropanes (23 . 31 min ) . Os- and trans enantiomers did not resolve. dards were used in co - injection experiments to determine the authenticity of P450 - catalyzed benzosultams. Authentic Designing enzymes that catalyze reactions not observed P450 catalyzed benzosultam samples were also prepared as in nature constitutes a contemporary challenge in protein described below and were characterized by NMR ( H and engineering (J . B . Siegel et al. , Science 329 , 309 ( 2010 )) . 40 13C ) and mass spectrometry . Working from a natural enzyme with promiscuous reactivity , Plasmids pCWori[ BM3] and pET22 were used as cloning this example demonstrates the construction of a cyclopro - vectors . Site -directed mutagenesis was accomplished by panase that exhibits kinetics comparable to natural enzymes, standard overlap mutagenesis using primers baring desired albeit with pre - activated reagents . Discovering catalysts for mutations ( IDT, San Diego , Calif. ) . Electro -competent non - natural bond -disconnections by screening natural 45 Escherichia coli cells were prepared following the protocol enzymes against synthetic reagents chosen based on chemi- of Sambrook et al. , Molecular cloning : a laboratory cal intuition offers a simple strategy for identifying enzymes manual. (Cold Spring Harbor Laboratory Press, New York , with basal levels of novel activity . As shown herein , a single 1989 ), vol. 2 . Restriction enzymes BamHI, EcoRI, Xhol, mutation can be enough to promote the new activity and Phusion polymerase , and T4 ligase were purchased from achieve synthetically useful stereoselectivities . The estab - 50 New England Biolabs ( NEB , Ipswich , Mass . ) . Alkaline lished reaction promiscuity of natural enzymes ( U . T . Born - phosphatase was obtained from Roche (Nutley , N . J . ) . The scheuer et al ., Angew . Chem . Int. Ed . 43 , 6032 ( 2004 ) ) and 1, 000x trace metal mix used in expression cultures con the ease with which cyclopropanase activity could be tained : 50 mM FeCl3 , 20 mM CaCl2, 10 mM MnSO4, 10 installed into P450BM3 indicates that this approach will be mM ZnSO4, 2 mM COS04, 2 mM CuCl2 , 2 mM NiCl2 , 2 useful for other synthetically important transformations for 55 mM Na M004, and 2 mM H2B03 . which biological counterparts do not yet exist . Enzyme Library Screening . Materials and Methods Libraries are stored at - 78° C . as glycerol stocks (Luria Unless otherwise noted , all chemicals and reagents for Bertani medium (LB amp ), 150 UL , 25 % v /v glycerol with 0 .1 chemical reactions were obtained from commercial suppli - mg/mL ampicillin ) in 96 -well plates. These stocks were used ers (Sigma -Aldrich , Acros ) and used without further puri- 60 to inoculate 96 - shallow -well plates containing 300 UL fication . The following heme proteins were all purchased LBm medium using a 96 - pin stamp . Single colonies from from Sigma -Aldrich : myoglobin ( from equine heart ) , per - site saturation libraries were picked with toothpicks and oxidase II ( from horseradish ), cytochrome c ( from bovine used to inoculate 300 uL of LB amp. The cells were incubated heart ) , catalase ( from Corynebacterium glutamicum ) and at 37° C . , 250 rpm , and 80 % relative humidity overnight . chloroperoxidase ( from Caldariomyces fumago ) . Silica gel 65 After 16 h , 50 uL aliquots of these over night cultures were chromatography purifications were carried out using AMD transferred into 2 mL, deep -well plates containing terrific Silica Gel 60 , 230 -400 mesh . 'H and 13C NMR spectra were broth ( TB amp) (800 uL containing 0 . 1 mg/ mL ampicillin , 1 US 10 , 208 , 322 B2 101 102 UL /mL trace metal mix and 20 mg L - aminolevulinic acid ) Thermostability Measurements . using a Multimek 96 - channel pipetting robot (Beckman Duplicate measurements were taken for all values Coulter , Fullerton , Calif. ). The cultures were incubated at reported on Tables 17 and 18 . Purified P450 solutions (4 uM , 37° C . for 3 h and 30 min , and 30 min after reducing the 200 uL ) were heated in a thermocycler ( Eppendorf) over a incubation temperature to 25° C . (250 rpm , 80 % relative 5 range of temperatures ( 38° C . -65° C . ) for 10 min followed humidity ) , 50 UL isopropyl B - D - 1 - thiogalactopyranoside by rapid cooling to 4° C . for 1 min . The precipitate was (IPTG , 4 .5 mM in TB amp) was added , and the cultures were removed by centrifugation . The concentration of folded allowed to continue for another 24 h at 25° C . ( 250 rpm , P450 remaining in the supernatant was measured by CO 80 % relative humidity ) . Cells were then pelleted ( 3 , 000xg , difference spectroscopy ( as described above ) . The tempera 15 min , 4° C . ) and stored at - 20° C . until further use , but at 10 ture at which half of the protein was denatured ( T30 ) was least for 2 h . For cell lysis , plates were allowed to thaw for determined by fitting the data to the equation : f ( T ) = 100 / ( 1 + 30 min at room temperature and then cell pellets were exp ( a * ( T - T5. ) ) ) . resuspended in 275 uL phosphate buffer ( 0 . 1 M , pH = 8 . 0 , Typical Procedure for Small - Scale Cyclopropanation Bio 0 .65 mg /mL lysozyme, 10 mM magnesium chloride and 40 conversions Under Anaerobic Conditions. U /mL DNAse I ). The lysing cells were incubated at 37° C . 15 Small - scale reactions ( 400 uL ) were conducted in 2 mL for 1 h . Cell debris was separated by centrifugation at crimp vials ( Agilent Technologies , San Diego , Calif . ). P450 5 ,000xg and 4° C . for 15 min . The resulting crude lysates solution (80 UL , 100 uM ) was added to the vial with a small were then transferred to 96 -well microtiter plates for CO stir bar before crimp sealing with a silicone septum . Phos assays and to 2 mL deep well plates for bioconversions. phate buffer ( 260 ul , 0 . 1 M , pH = 8 . 0 ) and 40 uL of a solution CO Binding Assay. 20 of the reductant ( 100 mM sodium dithionite , or 20 mM P450BM3 variants in cell lysate (40 uL ) were diluted with NADPH ) were combined in a larger crimp sealed vial and 60 UL phosphate buffer (0 . 1 M , pH = 8 . 0 ) . To this solution degassed by bubbling argon through the solution for at least was added 160 uL sodium dithionite ( 0 . 1 M in phosphate 5 min (FIG . 3 ) . In the meantime, the headspace of the 2 mL buffer , 0 . 1 M , pH = 8 . 0 ) . The absorbance at 450 and 490 nm reaction vial with the P450 solution was made anaerobic by was recorded using a Tecan M1000 UV /Vis plate reader, and 25 flushing argon over the protein solution ( with no bubbling ) . the microtiter plates were placed in a vacuum chamber . The When multiple reactions were conducted in parallel, up to 8 chamber was sealed , evacuated to approximately – 15 in Hg, reaction vials were degassed in series via cannula . The purged with CO gas, and incubated for 30 min . The plates buffer/ reductant solution ( 300 uL ) was syringed into the were then removed and the absorbance at 450 and 490 nm reaction vial, while under argon . The gas lines were discon was again recorded using a plate reader . The difference 30 nected from the reaction vial before placing the vials on a spectra could then be used to determine the P450 concen - plate stirrer . A 40x styrene solution in MeOH ( 10 UL , tration in each well as previously described ( C . R . Otey , in typically 1 . 2 M ) was added to the reaction vial via a glass Methods in Molecular Biology : Directed Enzyme Evolution , syringe , and left to stir for about 30 s . A 40xEDA solution F . H . Arnold , G . Georgiou , Eds. (Humana Press, Totowa, in MeOH was then added ( 10 uL , typically 400 mM ) and the N .J ., 2003 ) , vol. 230 ). 35 reaction was left stirring for the appropriate time. The final P450 Expression and Purification . concentrations of the reagents were typically : 30 mM sty For the enzymatic transformations, P450BM3 variants rene , 10 mM EDA , 10 mM sodium dithionite , 20 UM P450 . were used in purified form . Enzyme batches were prepared The reaction was quenched by adding 30 uL HCI (3M ) via as follows. One liter TBm was inoculated with an overnight syringe to the sealed reaction vial. The vials were opened culture ( 100 mL , LB mn ) of recombinant E . coli DH5a cells 40 and 20 ul internal standard (20 mM 2 -phenylethanol in harboring a pCWori plasmid encoding the P450 variant MeOH ) was added followed by 1 mL ethyl acetate . This under the control of the tac promoter. After 3 .5 h of mixture was transferred to a 1. 8 mL eppendorf tube which incubation at 37° C . and 250 rpm shaking (OD600 ca . 1 . 8 ) , was vortexed and centrifuged ( 16 ,000xg 1 min ) . The top the incubation temperature was reduced to 25° C . ( 30 min ), organic layer was dried over an anhydrous sodium sulfate and the cultures were induced by adding IPTG to a final 45 plug and analyzed by chiral phase GC . concentration of 0 . 5 mM . The cultures were allowed to A slightly modified work - up was implemented for kinetic continue for another 24 hours at this temperature . After experiments . The reactions were quenched after the set time harvesting the cells by centrifugation (4° C ., 15 min , 3, 000x by syringing 1 mL EtOAc to the closed vials and immedi g ) , the cell pellet was stored at - 20° C . until further use but ately vortexing themixture . The vials were then opened and at least for 2 h . The cell pellet was resuspended in 25 mM 50 20 uL internal standard was added . The mixture was trans Tris .HCl buffer (pH 7 . 5 at 25° C . ) and cells were lysed by ferred to a 1 . 8 mL eppendorf tube , vortexed and centrifuged sonication ( 2x1 min , output control 5 , 50 % duty cycle ; ( 16 ,000xg , 1 min ) . The top organic layer was dried over an Sonicator, Heat Systems — Ultrasonic , Inc . ) . Cell debris was anhydrous sodium sulfate plug and analyzed by GC . removed by centrifugation for 20 min at 4° C . and 27 ,000xg Typical Procedure for Preparative - Scale Cyclopropana and the supernatant was subjected to anion exchange chro - 55 tion Bioconversions Under Anaerobic Conditions. matography on a Q Sepharose column (HiTrapTM Q HP, GE The P450 solution was added to a Schlenk flask with a stir Healthcare, Piscataway , N . J .) using an AKTAxpress purifier bar. With the flask kept on ice, the head - space was evacuated FPLC system (GE healthcare ) . The P450 was eluted from and back - filled with argon (4x ) with care not to foam the the Q column by running a gradient from 0 to 0 . 5 M NaC1 protein solution . Phosphate buffer and reductant were pre over 10 column volumes (P450 elutes at 0 .35 M NaCl) . The 60 mixed and degassed together in a separate round -bottom P450 fractions were collected and concentrated using a 30 flask by bubbling argon through the solution for 20 min . The kDa molecular weight cut - off centrifugal filter and buffer - buffer / reductant solution was transferred to the Schlenk flask exchanged with 0 . 1 M phosphate buffer (pH = 8 . 0 ) . The via syringe . Styrene was added under argon and left to mix purified protein was flash - frozen on dry ice and stored at for 1 min . EDA was added dropwise under argon . The - 20° C . P450 concentration was determined in triplicate 65 solution was left to stir under argon until reaction comple using the CO binding assay described above ( 10 uL P450 tion . The reaction was quenched under argon by adding and 190 uL 0 . 1 M phosphate buffer , pH 8 . 0 , per well ) . hydrochloric acid ( 3 M ) to adjust the pH to 4 , before opening